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Sample records for high alkaline protease

  1. Production and Characterization of Alkaline Protease from a High Yielding and Moderately Halophilic Strain of SD11 Marine Bacteria

    Directory of Open Access Journals (Sweden)

    Hongxia Cui

    2015-01-01

    Full Text Available A marine bacterium SD11, which was isolated from sea muds (Geziwo Qinhuangdao Sea area, China, was used to produce thermostable alkaline serine nonmetal protease in the skim milk agar plate medium with 10% NaCl. The optimal temperature about the manufacture of the extracellular protease was ~60°C. The crude enzyme was stable at 20–50°C. The activity was retained to 60% and 45% after heating for 1 h at 60 and 70°C, respectively. The protease was highly active in a wide pH scope (8.0–10.0 and maximum protease activity exhibited at pH 10.0. The activity was restrained by phenylmethylsulfonyl fluoride (PMSF but mildly increased (~107% in the presence of ethylenediaminetetraacetic acid (EDTA, indicating that the production contains serine-protease(s and nonmetal protease(s. Moreover, the crude alkaline protease was active with the 5 mM Ca2+, Mn2+, Zn2+, Cu2+, Na+, and K+ that existed separately. In addition, the protease showed superduper stability when exposed to an anionic surfactant (5 mM SDS, an oxidizing agent (1% H2O2, and several organic solvents (methanol, isopropanol, and acetone. These results suggest that the marine bacterium SD11 is significant in the industry from the prospects of its ability to produce thermally stable alkaline protease.

  2. Effect of Proteolysis with Alkaline Protease Following High Hydrostatic Pressure Treatment on IgE Binding of Buckwheat Protein.

    Science.gov (United States)

    Lee, Chaeyoon; Lee, Wonhui; Han, Youngshin; Oh, Sangsuk

    2017-03-01

    Buckwheat is a popular food material in many Asian countries and it contains major allergenic proteins. This study was performed to analyze the effects of hydrolysis with alkaline protease following high hydrostatic pressure (HHP) treatment on the IgE binding of buckwheat protein. Extracted buckwheat protein was treated with HHP at 600 MPa for 30 min and hydrolyzed with alkaline protease for 240 min. IgE binding was examined using an enzyme-linked immunosorbent assay (ELISA) with serum samples from 14 patients who were allergic to buckwheat. Depending on the serum samples, HHP treatment of buckwheat protein without enzymatic hydrolysis decreased the IgE binding by 8.9% to 73.2% or increased by 31% to 78%. The IgE binding of buckwheat protein hydrolyzed with alkaline protease decreased by 73.8% to 100%. The IgE binding of buckwheat protein hydrolyzed with alkaline protease following HHP treatment decreased by 83.8% to 100%. This suggested that hydrolysis with alkaline protease following HHP treatment could be applied to reduce the IgE binding of buckwheat protein. © 2017 Institute of Food Technologists®.

  3. Thermodynamics of a Ca(2+)-dependent highly thermostable alkaline protease from a haloalkliphilic actinomycete.

    Science.gov (United States)

    Gohel, S D; Singh, S P

    2015-01-01

    An alkaline protease from salt-tolerant alkaliphilic actinomycetes, Nocardiopsis alba OK-5 was purified by a single-step hydrophobic interaction chromatography and characterized. The purified protease with an estimated molecular mass of 20 kDa was optimally active at 70 °C in 0-3 M NaCl and 0-100 mM Ca(2+) displaying significant stability at 50-80 °C. The enzyme was stable at 80 °C in 100 mM Ca(2+) with Kd of 17 × 10(-3) and t1/2 of 32 min. The activation energy (Ea), enthalpy (ΔH*), and entropy (ΔS*) for the protease deactivation calculated in the presence of 200 mM Ca(2+) were 38.15 kJ/mol, 35.49 kJ/mol and 183.48 J/mol, respectively. The change in free energy (ΔG*) for protease deactivation at 60 °C in 200 mM Ca(2+) was 95.88 kJ/mol. Decrease in ΔH* reflected reduced cooperativity of deactivation and unfolding. The enzyme was intrinsically stable that counteracted heat denaturation by a weak cooperativity during the unfolding. Further, the enzyme was highly stable in the presence of various cations, surfactants, H2O2, β-mercaptoethanol, and commercial detergents. The compatibility of the enzyme with various cations, surfactants, and detergent matrices suggests its suitability as an additive in the detergents and peptide synthesis.

  4. Crystal structure of the high-alkaline serine protease PB92 from Bacillus alcalophilus

    NARCIS (Netherlands)

    van der Laan, J.M.; Teplyakov, A.V.; Kelders, H.; Kalk, K.H.; Misset, O.; Mulleners, L.J.S.M.; Dijkstra, B.W.

    1992-01-01

    The crystal structure of a serine protease from the alkalophilic strain Bacillus alcalophilus PB92 has been determined by X-ray diffraction at 1.75 Å resolution. The structure has been solved by molecular replacement using the atomic model of subtilisin Carlsberg. The model of the PB92 protease has

  5. Alkaline Protease Production by a Strain of Marine Yeasts

    Institute of Scientific and Technical Information of China (English)

    WANG Ping; CHI Zhenming; MA Chunling

    2006-01-01

    Yeast strain 10 with high yield of protease was isolated from sediments of saltern near Qingdao, China.The protease had the highest activity at pH 9.0 and 45 ℃.The optimal medium for the maximum alkaline protease production of strain 10 was 2.5 g soluble starch and 2.0 g NaNO3 in 100 mL seawater with initial pH6.0.The optimal cultivation conditions for the maximum protease production were temperature 24.5 ℃, aeration rate 8.0 L min -1 and agitation speed 150 r min-1.Under the optimal conditions, 623.1 Umg-1 protein of alkaline protease was reached in the culture within 30 h of fermentation.

  6. Alkaline protease production by a strain of marine yeasts

    Science.gov (United States)

    Ping, Wang; Zhenming, Chi; Chunling, Ma

    2006-07-01

    Yeast strain 10 with high yield of protease was isolated from sediments of saltern near Qingdao, China. The protease had the highest activity at pH 9.0 and 45°C. The optimal medium for the maximum alkaline protease production of strain 10 was 2.5g soluble starch and 2.0g NaNO3 in 100mL seawater with initial pH 6.0. The optimal cultivation conditions for the maximum protease production were temperature 24.5°C, aeration rate 8.0L min-1 and agitation speed 150r min-1 Under the optimal conditions, 623.1 U mg-1 protein of alkaline protease was reached in the culture within 30h of fermentation.

  7. Isolation, identification and optimization of alkaline protease production by Candida viswanathii

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    Mandana Lotfi

    2014-03-01

    Conclusion: Due to the high demand for industrial enzymes in the Country and the high activity of alkaline proteases produced by strain. It seems that the native strain can achieve high production of alkaline proteases.These native strains could be resulted in the independence of our country in industrial enzymes production.

  8. Isolation of alkaline protease from Bacillus subtilis AKRS3

    African Journals Online (AJOL)

    ashok

    2012-08-28

    Aug 28, 2012 ... sodium chloride concentration), production by submerged fermentation and analytical ... subtilis AKRS3 for alkaline protease production indicated that 3% of .... using eight different carbon sources namely, glucose, lactose,.

  9. Stability and selectivity of alkaline proteases in hydrophilic solvents

    DEFF Research Database (Denmark)

    Pedersen, Lars Haastrup; Ritthitham, Sinthuwat; Pleissner, Daniel

    2008-01-01

    proteases, but at higher concentrations and particularly in anhydrous systems most enzymes including alkaline proteases will denature and consequently loose activity [1]. However, partial denaturing and increased structural flexibility due to the interaction between hydrophilic solvents and alkaline...... proteases has been agued as the primary reasons for increasing activity, influencing regio-selectivity and improving the enantio-selectivity of these enzymes [2]. Alkaline proteases have been shown to be active not only on peptides, but on a wide range of renewable resources for synthesis of biologically...... active molecules and carriers, and in synthesis of carbohydrate derivatives with designed functional properties.  When it comes to regio-selectivity of alkaline proateses on carbohydrates both the properties of the particular enzyme and the influence of the solvent is determining for the position...

  10. Alkaline protease production on date waste by an alkalophilic ...

    African Journals Online (AJOL)

    STORAGESEVER

    2008-05-16

    May 16, 2008 ... activity and stability towards anionic surfactants like SDS and oxidants ... Alkaline protease activity was determined by the method of. Higahara et al. ... rpm, the media was analyzed for cell mass, total protein concentration ...

  11. Optimization of alkaline protease production by Streptomyces sp ...

    African Journals Online (AJOL)

    Hacene

    2016-06-29

    Jun 29, 2016 ... The results showed the presence of an alkaline protease with optimal pH and ... significant effect on the production of the enzyme (fructose and malt extract), then defining theirs ..... Urease test. + ..... terminating inhibitors. Proc.

  12. PURIFICATION AND CHARACTERIZATION OF AN EXTRACELLULAR ALKALINE PROTEASE PRODUCED FROM AN ISOLATED BACILLUS SUBTILIS

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    Vijaya Bundela

    2013-03-01

    Full Text Available This paper describes the studies on the purification and partial characterization of serine alkaline protease produced through submerged fermentation process from a locally isolated Bacillus subtilis. This strain, grown in a highly alkaline medium (pH 10, produces an extracellular proteolytic enzyme. The alkaline protease was purified in a simple two-step procedure involving ammonium sulphate precipitation and gel filtration. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE analysis of the purified alkaline protease indicated an estimated molecular mass of 30KDa. It was more active in the range of 20-60ºC and had an optimum activity at 55ºC with optimum pH of 10.5. Characterization of the protease showed that it required certain cations such as Mg++, Mn++ and Ca++ for maximal activity. The serine nature of the alkaline protease was confirmed by PMSF inhibition. The temperature and pH stability of this Alkaline Protease from Bacillus Subtilismakes it potentially useful forindustrial applications.

  13. Alkaline protease production by solid state fermentation on polyurethane foam

    OpenAIRE

    Hongzhang, Chen; Hui, Wang; Aijun, Zhang; Zuohu, Li

    2006-01-01

    This paper investigated the process of solid state fermentation (SSF) using PUF (polyurethane foam) as inert solid support to produce alkaline protease. Maximal enzyme activity was 2185U/ml at pH 9.0, incubation temperature 32 0C inoculum amount of 1.0 % (v/v) , nutrient solution3.75 ml/g PUF, incubation time for 2 h and 15.0 mM of added CaCl2. Under the same conditions, the yield of alkaline protease produced by SSF using PUF as support is higher than that by submerged fermentation (SMF).

  14. Purification and Characterization of An Alkaline Protease from Acetes chinensis

    Institute of Scientific and Technical Information of China (English)

    XU Jiachao; LIU Xin; LI Zhaojie; XU Jie; XUE Changhu; GAO Xin

    2005-01-01

    An alkaline protease from Acetes chinensis was purified and characterized in this study. The steps of purification include ammonium sulfate precipitation, ion-exchange chromatography with Q-sepharose Fast Flow, gel filtration chromatography with S300 and the second ion-exchange chromatography with Q-sepharose Fast Flow. The protease was isolated and purified, which was present and active on protein substrates (azocasein and casein). The specific protease activity was 17.15folds and the recovery was 4.67. The molecular weight of the protease was estimated at 23.2 kD by SDS-PAGE. With azocasein as the susbstrate, the optimal temperature was 55 ℃ and the optimal pH value was 5.5. Ion Ca2+ could enhance the proteolytic activity of the protease, while Cu2+ , EDTA and PMSF could inhibit its activity.

  15. Purification and characterization of an alkaline protease from Acetes chinensis

    Science.gov (United States)

    Xu, Jiachao; Liu, Xin; Li, Zhaojie; Xu, Jie; Xue, Changhu; Gao, Xin

    2005-07-01

    An alkaline protease from Acetes chinensis was purified and characterized in this study. The steps of purification include ammonium sulfate precipitation, ion-exchange chromatography with Q-sepharose Fast Flow, gel filtration chromatography with S300 and the second ion-exchange chromatography with Q-sepharose Fast Flow. The protease was isolated and purified, which was present and active on protein substrates (azocasein and casein). The specific protease activity was 17.15 folds and the recovery was 4.67. The molecular weight of the protease was estimated at 23.2 kD by SDS-PAGE. With azocasein as the susbstrate, the optimal temperature was 55°C and the optimal pH value was 5.5. Ion Ca2+ could enhance the proteolytic activity of the protease, while Cu2+, EDTA and PMSF could inhibit its activity.

  16. Extraction and purification of a highly thermostable alkaline caseinolytic protease from wastes Penaeus vannamei suitable for food and detergent industries.

    Science.gov (United States)

    Dadshahi, Zahra; Homaei, Ahmad; Zeinali, Farrokhzad; Sajedi, Reza H; Khajeh, Khosro

    2016-07-01

    A novel thermostable protease was purified from Penaeus vannamei from Persian Gulf to homogeneity level using ammonium sulfate precipitation and anion-exchange chromatography. The purified protease showed a single band on native and SDS-PAGE with a molecular weight of 24kDa on SDS-PAGE. The enzyme showed the broad highest catalytic activity for hydrolysis of the substrate with maximal activity at pH 7 and 80°C. Activity of the enzyme was inhibited by Hg(2+), Zn(2+) Co(2+) and Cu(2+), while protease activity was increased in the presence of Fe(2+) and Mn(2+) by factors of 173% and 102%, respectively. Enzyme shows a broad substrate specificity and hydrolyzes both natural and synthetic substrates. Based on the Michaelis-Menten plots, the Km with casein as substrate was 16.8μM and Vmax was 82.6μM/min. The enzyme, derived from L. vannamei, possesses unique characteristics and could be used in various industrial and biotechnological applications.

  17. OPTIMIZATION OF MEDIA CONSTITUENTS FOR THE PRODUCTION OF ALKALINE PROTEASE FROM BACILLUS LICHENIFORMIS Mohideen

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    Mohideen Askar Nawas P

    2015-07-01

    Full Text Available Production of alkaline protease by Bacillus licheniformis has been investigated under submerged fermentation. The physical and chemical parameters influencing submerged fermentation were optimized. The effect of incubation time, temperature, pH, carbon sources and nitrogen sources and additional nutrients on the production of alkaline protease was characterized. The optimum conditions for the protease production by Bacillus licheniformis were found to be at pH 9.0 and temperature at 40ºC. The outcome of carbon and inorganic nitrogen sources on protease production proved that glucose and casein were the effective medium ingredients for Bacillus licheniformis respectively. The maximum amount of protease production was recorded in medium supplemented with ammonium sulphate. Among the tested metal ions, the level of protease yield was found to be high in medium supplemented with magnesium chloride. The protease production was amplified in the presence of 1.5% sodium chloride. The extreme stability towards Triton X-100, Tween 20 and SDS was observed in Bacillus licheniformis alkaline protease.

  18. Screening of Alkaline Protease-Producing Streptomyces diastaticus and Optimization of Enzyme Production

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    Elham Dawoodi

    2014-12-01

    Full Text Available Background and Aim: Alkaline proteases are used in pharmaceutical, film and photography, silk production and food, leather and detergent industries. Actinomycetes are gram positive bacteria that produce different enzymes such as proteases. The aims of this research were isolation of native alkaline protease-producing Actinomycete spp. from different soil samples as well as optimizing the conditions for enzyme production. Materials and Methods: The different soil samples were collected from different locations of the provinces of Khouzestan, Chahar Mahalo Bakhtiari and Isfahan, Iran. After determining of the best alkaline protease producing species using Lowry method, the optimization of alkaline protease was performed. Results: The alkaline protease producing Actinomycete spp. was isolated from soil. The most enzyme activity was measured in S.diastaticus. The best concentration of sucrose as the carbon source for the highest production of alkaline protease was 10 g/l. The optimum pH and temperature for the alkaline protease production by S. diastaticus were 10 and 30°C respectively. The maximum activity of alkaline protease was measured at 200 rpm as the best aeration speed. Conclusions: This is the first report of alkaline protease production by Streptomyces diastaticus in Iran. The accomplished examinations in this research confirmed the previous theories of alkaline protease production by Actinomycetes relatively. Regarding the immense applications of alkaline proteases in several industries and isolation of a native alkaline protease producing Actinomycete, The production potential of this enzyme in our country could be accessible in the near future.

  19. Alkaline protease from Thermoactinomyces sp. RS1 mitigates industrial pollution.

    Science.gov (United States)

    Verma, Amit; Ansari, Mohammad W; Anwar, Mohmmad S; Agrawal, Ruchi; Agrawal, Sanjeev

    2014-05-01

    Proteases have found a wide application in the several industrial processes, such as laundry detergents, protein recovery or solubilization, prion degradation, meat tenderizations, and in bating of hides and skins in leather industries. But the main hurdle in industrial application of proteases is their economical production on a large scale. The present investigation aimed to exploit the locally available inexpensive agricultural and household wastes for alkaline protease production using Thermoactinomyces sp. RS1 via solid-state fermentation (SSF) technique. The alkaline enzyme is potentially useful as an additive in commercial detergents to mitigate pollution load due to extensive use of caustic soda-based detergents. Thermoactinomyces sp. RS1 showed good protease production under SSF conditions of 55 °C, pH 9, and 50 % moisture content with potato peels as solid substrate. The presented findings revealed that crude alkaline protease produced by Thermoactinomyces sp. RS1 via SSF is of potential application in silver recovery from used X-ray films.

  20. Effect of amino acids on the repression of alkaline protease synthesis in haloalkaliphilic Nocardiopsis dassonvillei

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    Amit K. Sharma

    2016-12-01

    Full Text Available A newly isolated salt-tolerant alkaliphilic actinomycete, Nocardiopsis dassonvillei strain OK-18 grows on mineral salts medium with glucose as carbon source. It also grows and produces protease with amino acids as sole carbon source. The synthesis of extracellular alkaline protease parallel to growth was repressible by substrate concentrations. The absolute production of the protease was delinked with growth under nutritional stress, as protease production was high, despite poor growth. When amino acids served as the sole source of carbon and nitrogen, the enzyme production was significantly controlled by the number of amino acids. Maximal protease production was achieved with proline, asparagine, tyrosine, alanine, methionine and valine as sole source of carbon and nitrogen in minimal medium. With the increasing number of different amino acids in the presence and absence of glucose, the protease production was synergistically lower as compared to complex medium.

  1. Molecular characterization of alkaline protease of Bacillus amyloliquefaciens SP1 involved in biocontrol of Fusarium oxysporum.

    Science.gov (United States)

    Guleria, Shiwani; Walia, Abhishek; Chauhan, Anjali; Shirkot, C K

    2016-09-02

    An alkaline protease gene was amplified from genomic DNA of Bacillus amyloliquefaciens SP1 which was involved in effective biocontrol of Fusarium oxysporum. We investigated the antagonistic capacity of protease of B. amyloliquifaciens SP1, under in vitro conditions. The 5.62 fold purified enzyme with specific activity of 607.69U/mg reported 24.14% growth inhibition of F. oxysporum. However, no antagonistic activity was found after addition of protease inhibitor i.e. PMSF (15mM) to purified enzyme. An 1149bp nucleotide sequence of protease gene encoded 382 amino acids of 43kDa and calculated isoelectric point of 9.29. Analysis of deduced amino acid sequence revealed high homology (86%) with subtilisin E of Bacillus subtilis. The B. amyloliquefaciens SP1 protease gene was expressed in Escherichiax coli BL21. The expressed protease was secreted into culture medium by E. coli and exhibited optimum activity at pH8.0 and 60°C. The most reliable three dimensional structure of alkaline protease was determined using Phyre 2 server which was validated on the basis of Ramachandran plot and ERRAT value. The expression and structure prediction of the enzyme offers potential value for commercial application in agriculture and industry. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Enzymatic dehairing of goat skins using alkaline protease from Bacillus sp. SB12.

    Science.gov (United States)

    Briki, Selmen; Hamdi, Olfa; Landoulsi, Ahmed

    2016-05-01

    The present paper reports the production, purification and biochemical characterization of an extracellular alkaline protease from Bacillus sp. SB12. The enzyme has been used as an alternative to conventional chemicals treatment for dehairing of goat skins. The protease was optimally active at 37 °C and pH 9. Starch at 2% (w/v) was used as the best carbon source and the addition of yeast extract and peptone at 1% each supported the maximum level of protease production in the presence of 5 mM Ca(2+). Protease purification was performed with ammonium sulphate precipitation at 70% saturated fraction followed by dialysis and gel filtration chromatography using Sephadex G-100. The purified enzyme was homogeneous on non-denaturing PAGE and appeared as a single band with an apparent molecular weight of 41 kDa. This enzyme was moderately thermostable and has a wide pH stability range extending from pH 7 to 11. It showed high tolerance toward surfactants agents and organic solvents while it was completely inhibited by PMSF indicating the serine protease type. Purified protease was used to remove hair from goat skin proving its potential application in leather processing industry. The results revealed that the protease has enhanced the quality and physico-chemical properties of the skins while reducing the pollution.

  3. Purification and characterization of manganese-dependent alkaline serine protease from Bacillus pumilus TMS55.

    Science.gov (United States)

    Ibrahim, Kalibulla Syed; Muniyandi, Jeyaraj; Karutha Pandian, Shunmugiah

    2011-01-01

    The purification and characterization of a Mn2+-dependent alkaline serine protease produced by Bacillus pumilus TMS55 were investigated. The enzyme was purified in three steps: concentrating the crude enzyme using ammonium sulfate precipitation, followed by gel filtration and cation-exchange chromatography. The purified protease had a molecular mass of approximately 35 kDa, was highly active over a broad pH range of 7.0 to 12.0, and remained stable over a pH range of 7.5 to 11.5. The optimum temperature for the enzyme activity was found to be 60 degreesC. PMSF and AEBSF (1 mM) significantly inhibited the protease activity, indicating that the protease is a serine protease. Mn2+ ions enhanced the activity and stability of the enzyme. In addition, the purified protease remained stable with oxidants (H2O2, 2%) and organic solvents (25%), such as benzene, hexane, and toluene. Therefore, these characteristics of the protease and its dehairing ability indicate its potential for a wide range of commercial applications.

  4. Production of extracellular alkaline protease from Bacillus subtilis RSKK96 with solid state fermentation

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    Nurullah Akcan

    2011-09-01

    Full Text Available The production of extracellular alkaline protease by producing Bacillus subtilis RSKK96 was studied with solid state fermentation (SSF. Different agro residues as substrate were studied for enzyme production. The highest enzyme production was expressed with lentil husk as units per mass of dry substrate (3937.0 U/mg. Production parameters were optimized as incubation time 120 h, extraction medium Triton-X100 1%, initial moisture content 30%, initial pH 9.0. The high level of alkaline protease was obtained in the medium containing arabinose followed by lactose, galactose, and fructose. Among various nitrogen sources, beef extract was found to be the best inducer of alkaline protease, while other nitrogen sources repressed enzyme production. Among metal salts FeSO4.7H2O and MgSO4.7H2O was found to increase protease production. The maximum enzyme production (5759.2 U/mg was observed with lentil husk in 1000 mL of fermentation medium volume.

  5. A review on production of serine alkaline protease by Bacillus spp

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    Biswanath Bhunia

    2012-04-01

    Full Text Available Normal 0 false false false EN-US X-NONE X-NONE MicrosoftInternetExplorer4 In recent times, protease has gained considerable importance in the world market. Proteases are groups of proteins included in the subclass hydrolases, within the main class enzymes. Serine alkaline proteases (SAP are one of the most important groups of industrial enzymes. They account for approximately 35% of the total microbial enzyme sales. Serine protease is produced by various types of fermentation techniques using microorganism. Among the proteases, bacterial proteases are more significant than animal and fungal proteases. Bacillus is the most invigorated species producing extracellular proteases among many bacterial species that have found tremendous application in pharmaceutical, leather, laundry and food processing industry. Mathematical modeling of fermentation process helps understand the relationship between protease production and bacterial growth to provide quantitative information on the behavior of the system. Therefore, high level production of protease in industrial scale should be made feasible. The focus of the present review is to provide an updated overview of fermentative production and the factors that influence production, growth kinetics and downstream processing of serine alkaline protease. /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-qformat:yes; mso-style-parent:""; mso-padding-alt:0in 5.4pt 0in 5.4pt; mso-para-margin:0in; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:11.0pt; font-family:"Calibri","sans-serif"; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-fareast-font-family:"Times New Roman"; mso-fareast-theme-font:minor-fareast; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:"Times New Roman"; mso-bidi-theme-font:minor-bidi;}

  6. Purification and characterization of alkaline protease from Lysinibacillus fusiformis

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    Suppiah S*

    2012-08-01

    Full Text Available A novel alkaline protease producing bacterium was isolated from the gut of an estuarine fish Etroplus suratensis. The strain was identified by sequencing the fragment of their bacterial 16s rRNA and its homology was 97% closest to the Lysinibacillus fusiformis. An extracellular protease from this organism was purified by acetone precipitation, ion exchange chromatography and gel filtration chromatography methods and the specific activity of the purified enzyme was found to be 20.39 U/mg, 169.46U/mg and 352.0U/mg respectively. The molecular weight of the purified enzyme was determined to be 29kDa through SDS/PAGE analysis. The enzyme showed that the maximum at pH 9.0 and temperature at 40ºC. The purified enzyme remains active in the presence of various metal ions and it was strongly stimulated by the addition of Ca2+. Among the tested surfactants, the optimum activity was observed in SDS when compared to the other tested surfactants. Normal 0 false false false EN-US X-NONE X-NONE

  7. Digestive alkaline proteases from thornback ray (Raja clavata): Characteristics and applications.

    Science.gov (United States)

    Lassoued, Imen; Hajji, Sawssen; Mhamdi, Samiha; Jridi, Mourad; Bayoudh, Ahmed; Barkia, Ahmed; Nasri, Moncef

    2015-09-01

    This study describes the characterization of a crude protease extract from thornback ray (Raja clavata) and its evaluation in liquid detergent and in deproteinizattion of shrimp waste. At least five clear caseinolytic proteases bands were observed in a zymogram. The crude protease showed optimum activity at pH 8.0 and 50 °C, and it was highly stable over pH range from 8.0 to 11.0. Proteolytic enzymes were very stable in non-ionic surfactants and in the presence of oxidizing agents, maintaining 70% of their activity after incubation for 1 h at 30 °C in the presence of 1% sodium perborate. In addition, they showed high stability and compatibility with various liquid laundry-detergents available in the Tunisian market. The crude extract retained 100% of its activity after preincubation for 60 min at 30 °C in the presence of Nadhif Perfect, Textil and Carrefour laundry detergents. Further, proteases from R. clavata viscera were used for shrimp waste deproteinization in the process of chitin preparation. The percent of protein removal after 3 h hydrolysis at 45 °C with an enzyme/substrate ratio of 30 U/mg of proteins was 74%. These results suggest that enzymatic deproteinization of shrimp wastes by fish endogenous alkaline proteases could be applicable to the chitin production process. Copyright © 2015 Elsevier B.V. All rights reserved.

  8. Digestive Alkaline Proteases from Zosterisessor ophiocephalus, Raja clavata, and Scorpaena scrofa: Characteristics and Application in Chitin Extraction.

    Science.gov (United States)

    Nasri, Rim; Younes, Islem; Lassoued, Imen; Ghorbel, Sofiane; Ghorbel-Bellaaj, Olfa; Nasri, Moncef

    2011-01-01

    The aim of this work was to study some biochemical characteristics of crude alkaline protease extracts from the viscera of goby (Zosterisessor ophiocephalus), thornback ray (Raja clavata), and scorpionfish (Scorpaena scrofa), and to investigate their applications in the deproteinization of shrimp wastes. At least four caseinolytic proteases bands were observed in zymogram of each enzyme preparation. The optimum pH for enzymatic extracts activities of Z. ophiocephalus, R. clavata, and S. scrofa were 8.0-9.0, 8.0, and 10.0, respectively. Interestingly, all the enzyme preparations were highly stable over a wide range of pH from 6.0 to 11.0. The optimum temperatures for enzyme activity were 50°C for Z. ophiocephalus and R. clavata and 55°C for S. scrofa crude alkaline proteases. Proteolytic enzymes showed high stability towards non-ionic surfactants (5% Tween 20, Tween 80, and Triton X-100). In addition, crude proteases of S. scrofa, R. clavata, and Z. ophiocephalus were found to be highly stable towards oxidizing agents, retaining 100%, 70%, and 66%, respectively, of their initial activity after incubation for 1 h in the presence of 1% sodium perborate. They were, however, highly affected by the anionic surfactant SDS. The crude alkaline proteases were tested for the deproteinization of shrimp waste in the preparation of chitin. All proteases were found to be effective in the deproteinization of shrimp waste. The protein removals after 3 h of hydrolysis at 45°C with an enzyme/substrate ratio (E/S) of 10 were about 76%, 76%, and 80%, for Z. ophiocephalus, R. clavata, and S. scrofa crude proteases, respectively. These results suggest that enzymatic deproteinization of shrimp wastes by fish endogenous alkaline proteases could be applicable to the chitin production process.

  9. Stability and selectivity of alkaline proteases in hydrophilic solvents

    DEFF Research Database (Denmark)

    Pedersen, Lars Haastrup; Ritthitham, Sinthuwat; Pleissner, Daniel

    2008-01-01

    were synthesised in anhydrous DMF and pyridine, respectively with different acyl donors and a number of different subtilisins as biocatalysts - in all cases the 1'-O-monoester was the major product [6,7,8,9]. But the alkaline protease AL89 revealed regio-selectivity towards the C-2 position of sucrose....... Shibatani, Y. Maekawa, Y. Hiraguri, R. Kurane, Y. Tokiwa, Biotechnol. Lett.1999, 21, 355. [4]           S. Riva, J. Chopineau, A.P.G. Kieboom, A. Klibanov, J. Am. Chem. Soc.,1988, 110, 584. [5]           T. Watanabe, R. Matsue, Y. Honda, M. Kuwahara, Carbohydr. Res., 1995, 275, 215. [6]           P. Potier......, A. Bouchu, G. Descotes, Y. Queneau, Tetrahedron: Lett. 2000, 41, 3597. [7]           Q. Wu, N. Wang, Y.M. Xiao, D.S. Lu, X.F. Lin, Carbohydr. Res., 2004, 339, 2059. [8]           H.G. Park, H.N. Chang, Biotechnol. Lett. 2000, 22, 39 [9]           S. Riva, M. Nonini, G. Ottolina, B. Danieli...

  10. Purification and characterization of alkaline protease from Alcaligenes faecalis.

    Science.gov (United States)

    Thangam, E Berla; Rajkumar, G Suseela

    2002-04-01

    Extracellular alkaline protease from the alkalophilic bacterium Alcaligenes faecalis was purified by a combination of ion-exchange and size-exclusion chromatographic methods, and its properties were examined. The purified enzyme had a specific activity of 563.8 micromol of tyrosine/min per mg of protein and gave a single band on native PAGE and SDS/PAGE with a molecular mass of 67 kDa. Gelatin zymogram also revealed one clear zone of proteolytic activity which corresponded to the band obtained with native PAGE and SDS/PAGE. The enzyme had an optimal pH of 9.0 and exhibited its highest activity at 55 degrees C. The enzyme activity was inhibited by PMSF, suggesting the presence of serine residues at the active site. The enzyme had a K(m) of 1.66 mg/ml and a V(max) of 526 units/min per mg of protein with casein as the substrate.

  11. Regiospecific Addition of Uracil to Acrylates Catalyzed by Alkaline Protease from Bacillus subtilis

    Institute of Scientific and Technical Information of China (English)

    Ying CAI; Jian Yi WU; Na WANG; Xiao Feng SUN; Xian Fu LIN

    2004-01-01

    Michael addition reactions of uracil to acrylates were catalyzed by an alkaline protease from Bacillus subtilis in dimethyl sulfoxide at 55 ℃ for 72 h. The adducts were determined by TLC, IR and 1H NMR.

  12. Statistical optimization of alkaline protease production from Penicillium citrinum YL-1 under solid-state fermentation.

    Science.gov (United States)

    Xiao, Yun-Zhu; Wu, Duan-Kai; Zhao, Si-Yang; Lin, Wei-Min; Gao, Xiang-Yang

    2015-01-01

    Proteases from halotolerant and halophilic microorganisms were found in traditional Chinese fish sauce. In this study, 30 fungi were isolated from fermented fish sauce in five growth media based on their morphology. However, only one strain, YL-1, which was identified as Penicillium citrinum by internal transcribed spacer (ITS) sequence analysis, can produce alkaline protease. This study is the first to report that a protease-producing fungus strain was isolated and identified in traditional Chinese fish sauce. Furthermore, the culture conditions of alkaline protease production by P. citrinum YL-1 in solid-state fermentation were optimized by response surface methodology. First, three variables including peptone, initial pH, and moisture content were selected by Plackett-Burman design as the significant variables for alkaline protease production. The Box-Behnken design was then adopted to further investigate the interaction effects between the three variables on alkaline protease production and determine the optimal values of the variables. The maximal production (94.30 U/mL) of alkaline protease by P. citrinum YL-1 took place under the optimal conditions of peptone, initial pH, and moisture content (v/w) of 35.5 g/L, 7.73, and 136%, respectively.

  13. Deep-sea fungi as a source of alkaline and cold-tolerant proteases

    Digital Repository Service at National Institute of Oceanography (India)

    Damare, S.; Raghukumar, C.; Muraleedharan, U.; Raghukumar, S.

    and Chang CS. Bleach resistant alkaline protease produced by a Bacillus sp. Isolated from the Korean polychaete, Periserrula leucophryna. Proc Biochem 2004;39:1441-7. [12] Turkiewicz M, Gromek E, Kalinowska H and Zielinska M, Biosynthesis and properties... o C Serine protease [33] Paecilomyces lilacinus Biocontrol agent Azocasein [34] Bacillus sp Korean polychaete Casein 1?g tyrosine min -1 10.0 45-50 o C Serine protease [11] Aspergillus ustus NIOCC #20 Deep-sea sediment Azocasein 1639 ACU m...

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

    Science.gov (United States)

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

    2014-01-01

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

  15. A metagenomic alkaline protease from saline habitat: cloning, over-expression and functional attributes.

    Science.gov (United States)

    Purohit, Megha K; Singh, Satya P

    2013-02-01

    Metagenomics has opened new horizon to unlock the biotechnological potential for novel enzymes. An alkaline protease gene was obtained from the total environmental DNA extracted from a saline habitat. After cloning and sequencing, it was identified that the protease gene related to uncultivable bacteria (HM219181). The protease was over expressed at 6h of induction with optimum induction at 1mM IPTG and 27°C. The purified enzyme was characterized with respect to various factors; temperature, pH, NaCl and chemical denaturant. The sequence analysis indicated a hydrophobic tendency of the protein, while the predicted 3D structure indicated the enzyme as a serine protease.

  16. Expression and characterization of Coprothermobacter proteolyticus alkaline serine protease

    Science.gov (United States)

    TECHNICAL ABSTRACT A putative protease gene (aprE) from the thermophilic bacterium Coprothermobacter proteolyticus was cloned and expressed in Bacillus subtilis. The enzyme was determined to be a serine protease based on inhibition by PMSF. Biochemical characterization demonstrated the enzyme had...

  17. Purification and characterization of an alkaline protease from Micrococcus sp. isolated from the South China Sea

    Science.gov (United States)

    Hou, Enling; Xia, Tao; Zhang, Zhaohui; Mao, Xiangzhao

    2017-04-01

    Protease is wildly used in various fields, such as food, medicine, washing, leather, cosmetics and other industrial fields. In this study, an alkaline protease secreted by Micrococcus NH54PC02 isolated from the South China Sea was purified and characterized. The growth curve and enzyme activity curve indicated that the cell reached a maximum concentration at the 30th hour and the enzyme activity reached the maximum value at the 36th hour. The protease was purified with 3 steps involving ammonium sulfate precipitation, ion-exchange chromatography and hydrophobic chromatography with 8.22-fold increase in specific activity and 23.68% increase in the recovery. The molecular mass of the protease was estimated to be 25 kDa by SDS-PAGE analysis. The optimum temperature and pH for the protease activity were 50°C and pH 10.0, respectively. The protease showed a strong stability in a wide range of pH values ranging from 6.0-11.0, and maintained 90% enzyme activity in strong alkaline environment with pH 11.0. Inhibitor trials indicated that the protease might be serine protease. But it also possessed the characteristic of metalloprotease as it could be strongly inhibited by EDTA and strongly stimulated by Mn2+. Evaluation of matrix-assisted laser desorption ionization/time-of-flight MS (MALDI-TOF-TOF/MS) showed that the protease might belong to the peptidase S8 family.

  18. Kinetic study of alkaline protease 894 for the hydrolysis of the pearl oyster Pinctada martensii

    Institute of Scientific and Technical Information of China (English)

    CHEN Xin; CHEN Hua; CAI Bingna; LIU Qingqin; SUN Huili

    2013-01-01

    A new enzyme (alkaline protease 894) obtained from the marine extremophile Flavobacterium yellowsea (YS-80-122) has exhibited strong substrate-binding and catalytic activity,even at low temperature,but the characteristics of the hydrolysis with this enzyme are still unclear.The pearl oyster Pinctada martensii was used in this study as the raw material to illustrate the kinetic properties ofprotease 894.After investigating the intrinsic relationship between the degree of hydrolysis and several factors,including initial reaction pH,temperature,substrate concentration,enzyme concentration,and hydrolysis time,the kinetics model was established.This study showed that the optimal conditions for the enzymatic hydrolysis were an initial reaction pH of 5.0,temperature of 30℃,substrate concentration of 10% (w/v),enzyme concentration of 2 500 U/g,and hydrolysis time of 160 min.The kinetic characteristics of the protease for the hydrolysis of P.martensii were obtained.The inactivation constant was found to be 15.16/min,and the average relative error between the derived kinetics model and the actual measurement was only 3.04%,which indicated a high degree of fitness.Therefore,this study provides a basis for the investigation of the concrete kinetic characteristics of the new protease,which has potential applications in the food industry.

  19. Screening of Strains Producing Alkaline Protease from Soil and Study on the Conditions for Enzyme Production

    Institute of Scientific and Technical Information of China (English)

    2011-01-01

    [Objective] The aim was to screen strains producing alkaline protease from soil and study the conditions for enzyme production.[Method] Eight strains producing alkaline protease were isolated from soil through plate isolation,and the ability of enzyme production was measured by filter paper and Folin-phenol method.The strain with the strongest ability of enzyme production was screened as a candidate strain,then the factors influencing the ability of enzyme production was studied,finally the conditions for e...

  20. Efficient proteolysis and application of an alkaline protease from halophilic Bacillus sp. EMB9.

    Science.gov (United States)

    Sinha, Rajeshwari; Srivastava, A K; Khare, S K

    2014-10-03

    A salt-stable alkaline protease from moderately halophilic Bacillus sp. EMB9, isolated from the western coast of India, is described. This protease was capable of efficiently removing silver from used/waste X-Ray films, as well as hydrolyzing defatted soy flour with 31% degree of hydrolysis (DH). Production of the protease was optimized by using response surface methodology. Ca(2+) and NaCl were the most critical factors in enhancing the yield. Under optimized culture conditions, a maximum of 369 U protease/mL was obtained, which is quite comparable to the yields of commercial proteases. The elevated production level coupled with ability to efficiently hydrolyze protein-laden soy flour and complete recovery of silver from used X-Ray films makes it a prospective industrial enzyme.

  1. Purification and properties of detergent-compatible extracellular alkaline protease from Scopulariopsis spp.

    Science.gov (United States)

    Niyonzima, Francois Niyongabo; More, Sunil

    2014-10-03

    A fungal alkaline protease of Scopulariopsis spp. was purified to homogeneity with a recovery of 32.2% and 138.1 U/mg specific activity on lectin-agarose column. The apparent molecular mass was 15 ± 1 kD by sodium dodecyl sulfate polyacryalamide gel electrophoresis (SDS-PAGE). It was a homogenous monomeric glycoprotein as shown by a single band and confirmed by native PAGE and gelatin zymography. The enzyme was active and stable over pH range 8.0-12.0 with optimum activity at pH 9.0. The maximum activity was recorded at 50°C and remained unaltered at 50°C for 24 hr. The enzyme was stimulated by Co(2+) and Mn(2+) at 10 mM but was unaffected by Ba(2+), Mg(2+), Cu(2+), Na(+), K(+), and Fe(2+). Ca(2+) and Fe(3+) moderately reduced the activity (∼18%); however, a reduction of about 40% was seen for Zn(2+) and Hg(2+). The enzyme activity was completely inhibited by 5 mM phenylmethylsulfonyl fluoride (PMSF) and partially by N-bromosuccinimide (NBS) and tocylchloride methylketone (TLCK). The serine, tryptophan, and histidine may therefore be at or near the active site of the enzyme. The protease was more active against gelatin compared to casein, fibrinogen, egg albumin, and bovine serum albumin (BSA). With casein as substrate, Km and Vmax were 4.3 mg/mL and 15.9 U/mL, respectively. An activation was observed with sodium dodecyl sulfate (SDS), Tween-80, and Triton X-100 at 2% (v/v); however, H2O2 and NaClO did not affect the protease activity. Storage stability was better for all the temperatures tested (-20, 4, and 28 ± 2°C) with a retention of more than 85% of initial activity after 40 days. The protease retained more than 50% activity after 24 hr of incubation at 28, 60, and 90°C in the presence (0.7%, w/v) of commercial enzymatic and nonenzymatic detergents. The Super Wheel-enzyme solution was able to completely remove blood staining, differing from the detergent solution alone. The stability at alkaline pH and high temperatures, broad substrate specificity

  2. An overview on fermentation, downstream processing and properties of microbial alkaline proteases.

    Science.gov (United States)

    Gupta, R; Beg, Q K; Khan, S; Chauhan, B

    2002-12-01

    Microbial alkaline proteases dominate the worldwide enzyme market, accounting for a two-thirds share of the detergent industry. Although protease production is an inherent property of all organisms, only those microbes that produce a substantial amount of extracellular protease have been exploited commercially. Of these, strains of Bacillus sp. dominate the industrial sector. To develop an efficient enzyme-based process for the industry, prior knowledge of various fermentation parameters, purification strategies and properties of the biocatalyst is of utmost importance. Besides these, the method of measurement of proteolytic potential, the selection of the substrate and the assay protocol depends upon the ultimate industrial application. A large array of assay protocols are available in the literature; however, with the predominance of molecular approaches for the generation of better biocatalysts, the search for newer substrates and assay protocols that can be conducted at micro/nano-scale are becoming important. Fermentation of proteases is regulated by varying the C/N ratio and can be scaled-up using fed-batch, continuous or chemostat approaches by prolonging the stationary phase of the culture. The conventional purification strategy employed, involving e.g., concentration, chromatographic steps, or aqueous two-phase systems, depends on the properties of the protease in question. Alkaline proteases useful for detergent applications are mostly active in the pH range 8-12 and at temperatures between 50 and 70 degrees C, with a few exceptions of extreme pH optima up to pH 13 and activity at temperatures up to 80-90 degrees C. Alkaline proteases mostly have their isoelectric points near to their pH optimum in the range of 8-11. Several industrially important proteases have been subjected to crystallization to extensively study their molecular homology and three-dimensional structures.

  3. Regioselective Synthesis of Polymerizable Vinyl Guaifenesin Esters Catalyzed by an Alkaline Protease of Bacillus subtilis

    Institute of Scientific and Technical Information of China (English)

    Na WANG; Qi WU; Jian Ming XU; Xiu Ming JIANG; Xian Fu LIN

    2004-01-01

    Three polymerizable vinyl guaifenesin esters with different acyl donor carbon chain lengths (C4,C6,C10) were regioselectivly synthesized by an alkaline protease from Bacillus subtilis in pyridine at 50°C for 1, 3, 5 days respectively.

  4. Enhanced production of alkaline protease by a mutant of Bacillus licheniformis N-2 for dehairing

    Directory of Open Access Journals (Sweden)

    Muhammad Nadeem

    2010-10-01

    Full Text Available The purpose of the present investigations was to improve the yield of alkaline protease for leather dehairing by subjecting the indigenous proteolytic strain Bacillus licheniformis N-2 to various mutagenic treatments viz. UV irradiations, NTG (N-methyl-N-nitro-N-nitrosoguinidine and MMS (methyl methane sulfonate. After screening on skim milk agar plates, a total of nine positive mutants were selected for shake flask experiments. Among these, the best proteolytic mutant designated as UV-9 showed 1.4 fold higher alkaline protease activity in preoptimized growth medium than the parent strain. The fermentation profile and kinetic parameters such u(h-1, Yp/s, Yp/x, Yx/s, q s, Qs, q p and Qp also indicated the superiority of the selected mutant UV-9 for alkaline protease production over the parent strain and rest of the mutants. The dehairing capability of mutant UV-9 alkaline protease was analyzed by soaking goat skin pieces for different time intervals (3-15 h at 40 º C. A complete dehairing without degradation of collagen was achieved after 12 h, indicating its commercial exploitation in leather industry.

  5. Engineering of quorum-sensing systems for improved production of alkaline protease by Bacillus subtilis

    NARCIS (Netherlands)

    Tjalsma, H; Koetje, EJ; Kiewiet, R; Kuipers, OP; Kolkman, M; van der Laan, J; Daskin, R; Ferrari, E; Bron, S

    2004-01-01

    Aim: Engineering of Rap-Phr quorum-sensing systems of Bacillus subtilis and subsequent evaluation of the transcription of the aprE gene, encoding a major extracellular alkaline protease. Methods and Results: Addition of synthetic Phr pentapeptides to the growth medium, or overproduction of pre-Phr p

  6. Purification and characterization of a novel extracellular alkaline protease from Cellulomonas bogoriensis.

    Science.gov (United States)

    Li, Fan; Yang, Liyuan; Lv, Xue; Liu, Dongbo; Xia, Hongmei; Chen, Shan

    2016-05-01

    An extracellular alkaline protease produced by the alkali-tolerant Cellulomonas bogoriensis was purified by a combination of ammonium sulfate precipitation and cation exchange chromatography. The purity of the protease was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and its molecular weight was confirmed to be 18.3 kDa. The enzyme showed optimum activity at 60 °C and pH 11. The stability of the protease was maintained at a wide temperature range of 4-60 °C and pH range of 3-12. Irreversible inhibition of the enzyme activity by phenylmethylsulfonyl fluoride and tosyl-l-phenylalanine chloromethyl ketone demonstrated that the purified enzyme is a chymotrypsin of the serine protease family. The Km and Vmax of the protease activity on casein were 19.2 mg/mL and 25000 μg/min/mg, respectively. The broad substrate specificity and remarkable stability in the presence of organic solvents, salt, and commercial detergents, as well as its excellent stain removal and dehairing capability, make the purified alkaline protease a promising candidate for industrial applications.

  7. Molecular cloning, sequence and structural analysis of dehairing Mn(2+) dependent alkaline serine protease (MASPT) of Bacillus pumilus TMS55.

    Science.gov (United States)

    Ibrahim, Kalibulla Syed; Muniyandi, Jeyaraj; Pandian, Shunmugiah Karutha

    2011-10-01

    Leather industries release a large amount of pollution-causing chemicals which creates one of the major industrial pollutions. The development of enzyme based processes as a potent alternative to pollution-causing chemicals is useful to overcome this issue. Proteases are enzymes which have extensive applications in leather processing and in several bioremediation processes due to their high alkaline protease activity and dehairing efficacy. In the present study, we report cloning, characterization of a Mn2+ dependent alkaline serine protease gene (MASPT) of Bacillus pumilus TMS55. The gene encoding the protease from B. pumilus TMS55 was cloned and its nucleotide sequence was determined. This gene has an open reading frame (ORF) of 1,149 bp that encodes a polypeptide of 383 amino acid residues. Our analysis showed that this polypeptide is composed of 29 residues N-terminal signal peptide, a propeptide of 79 residues and a mature protein of 275 amino acids. We performed bioinformatics analysis to compare MASPT enzyme with other proteases. Homology modeling was employed to model three dimensional structure for MASPT. Structural analysis showed that MASPT structure is composed of nine α-helices and nine β-strands. It has 3 catalytic residues and 14 metal binding residues. Docking analysis showed that residues S223, A260, N263, T328 and S329 interact with Mn2+. This study allows initial inferences about the structure of the protease and will allow the rational design of its derivatives for structure-function studies and also for further improvement of the enzyme.

  8. Purification and characterization of cloned alkaline protease gene of Geobacillus stearothermophilus.

    Science.gov (United States)

    Iqbal, Irfana; Aftab, Muhammad Nauman; Afzal, Mohammed; Ur-Rehman, Asad; Aftab, Saima; Zafar, Asma; Ud-Din, Zia; Khuharo, Ateeque Rahman; Iqbal, Jawad; Ul-Haq, Ikram

    2015-02-01

    Thermostable alkaline serine protease gene of Geobacillus stearothermophilus B-1172 was cloned and expressed in Escherichia coli BL21 (DE3) using pET-22b(+), as an expression vector. The growth conditions were optimized for maximal production of the protease using variable fermentation parameters, i.e., pH, temperature, and addition of an inducer. Protease, thus produced, was purified by ammonium sulfate precipitation followed by ion exchange chromatography with 13.7-fold purification, with specific activity of 97.5 U mg(-1) , and a recovery of 23.6%. Molecular weight of the purified protease, 39 kDa, was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme was stable at 90 °C at pH 9. The enzyme activity was steady in the presence of EDTA indicating that the protease was not a metalloprotease. No significant change in the activity of protease after addition of various metal ions further strengthened this fact. However, an addition of 1% Triton X-100 or SDS surfactants constrained the enzyme specific activity to 34 and 19%, respectively. Among organic solvents, an addition of 1-butanol (20%) augmented the enzyme activity by 29% of the original activity. With casein as a substrate, the enzyme activity under optimized conditions was found to be 73.8 U mg(-1) . The effect of protease expression on the host cells growth was also studied and found to negatively affect E. coli cells to certain extent. Catalytic domains of serine proteases from eight important thermostable organisms were analyzed through WebLogo and found to be conserved in all serine protease sequences suggesting that protease of G. stearothermophilus could be beneficially used as a biocontrol agent and in many industries including detergent industry.

  9. Effect of Medium Composition on Commercially Important Alkaline Protease Production by Bacillus licheniformis N-2

    Directory of Open Access Journals (Sweden)

    Javed Iqbal Qazi

    2008-01-01

    Full Text Available Protease production by alkalophilic B. licheniformis N-2 was investigated in 50 mL of the growth medium consisting of (in g/L: glucose 10.0, soybean meal 10.0, K2HPO4 3.0, MgSO4·7H2O 0.5, NaCl 0.5 and CaCl2·2H2O 0.5 at pH=10. Different carbon and nitrogen sources in the form of fine powder of organic, inorganic and defatted meals were studied to select the suitable substrate for alkaline protease production. The highest level of alkaline protease (677.64 U/mL was obtained in the medium containing glucose followed by soluble starch and wheat bran. Among various nitrogen sources, defatted soybean meal was found to be the best inducer of alkaline protease, while inorganic nitrogen sources in the form of ammonium salts repressed the enzyme activity up to 96 %. Thermostability studies showed that the enzyme in the presence of 10 mM Ca2+ ions retained its residual activity up to 80 % even after incubation at 40 °C for 12 h. The enzyme was found stable over a broad range of pH (8–11 and lost 52 % of its residual activity at pH=12. After the treatment with Tween 20, Tween 45, Tween 65, Triton X-405, H2O2 and sodium perborate, each at 1.0 % concentration, the enzyme showed residual activity of 105, 82, 116, 109, 135 and 126 %, respectively. The application of alkaline protease for removal of blood stains from cotton fabric also indicates its potential use in detergent formulations.

  10. Development of novel robust nanobiocatalyst for detergents formulations and the other applications of alkaline protease.

    Science.gov (United States)

    Ibrahim, Abdelnasser S S; El-Toni, Ahmed M; Al-Salamah, Ali A; Almaary, Khalid S; El-Tayeb, Mohamed A; Elbadawi, Yahya B; Antranikian, Garabed

    2016-05-01

    Alkaline protease from alkaliphilic Bacillus sp. NPST-AK15 was immobilized onto functionalized and non-functionalized rattle-type magnetic core@mesoporous shell silica (RT-MCMSS) nanoparticles by physical adsorption and covalent attachment. However, the covalent attachment approach was superior for NPST-AK15 protease immobilization onto the activated RT-MCMSS-NH₂nanoparticles and was used for further studies. In comparison to free protease, the immobilized enzyme exhibited a shift in the optimal temperature and pH from 60 to 65 °C and pH 10.5-11.0, respectively. While free protease was completely inactivated after treatment for 1 h at 60 °C, the immobilized enzyme maintained 66.5% of its initial activity at similar conditions. The immobilized protease showed higher k cat and K m , than the soluble enzyme by about 1.3-, and 1.2-fold, respectively. In addition, the results revealed significant improvement of NPST-AK15 protease stability in variety of organic solvents, surfactants, and commercial laundry detergents, upon immobilization onto activated RT-MCMSS-NH₂nanoparticles. Importantly, the immobilized protease maintained significant catalytic efficiency for ten consecutive reaction cycles, and was separated easily from the reaction mixture using an external magnetic field. To the best of our knowledge this is the first report about protease immobilization onto rattle-type magnetic core@mesoporous shell silica nanoparticles that also defied activity-stability tradeoff. The results clearly suggest that the developed immobilized enzyme system is a promising nanobiocatalyst for various bioprocess applications requiring a protease.

  11. Production and some properties of crude alkaline proteases of indigenous Central Amazonian rhizobia strains

    Directory of Open Access Journals (Sweden)

    Arlem Nascimento de Oliveira

    2010-10-01

    Full Text Available Two rhizobia strains isolated from soils of the Central Amazonian floodplain produced appreciable quantities of crude alkaline protease extracts with inexpensive carbon and nitrogen sources. These protease crude extracts were optimally active at pH 9.0-11.0. The optimum temperatures were 35 ºC for Rhizobium sp. strain R-986 and 55 ºC for Bradyrhizobium sp. strain R-993. Protease activities in the crude extracts were enhanced in the presence of 5 mM metal ions, such as Na+, Ca2+, Mg2+ and Mn2+. Rhizobia proteases were strongly inhibited by PMSF, a serine-protease inhibitor. The enzymes were active in the presence of surfactants (SDS and Triton X-100 and stable in oxidizing (H2O2 and reducing agents (β-mercaptoethanol, and organic solvents (acetone, hexane, methanol, 1-propanol and toluene.Duas estirpes de rizóbia isoladas de solos de várzea da Amazônia Central produziram grandes quantidades de proteases alcalinas extracelulares, usando fontes baratas de carbono e nitrogênio. Os extratos brutos de proteases foram ativos em pH 9,0-11,0. As temperaturas ótimas foram de 35 ºC para a enzima do Rhizobium R-986 e de 55 ºC para a do Bradyrhizobium R-993. As atividades proteolíticas aumentaram na presença de 5 mM dos íons Na+, Ca2+ , Mg2+ e Mn2+ . As proteases secretadas pelos rizóbios foram fortemente inibidas por PMSF, um inibidor de serina protease. As enzimas foram ativas na presença de surfactantes (SDS e Triton X-100, e estáveis na presença de agentes oxidantes (H2O2 e redutores (β-mercaptoetanol e solventes orgânicos (acetona, hexano, metanol, 1-propanol e tolueno.

  12. Advances on microbial thermophilic alkaline protease%微生物高温碱性蛋白酶

    Institute of Scientific and Technical Information of China (English)

    陈杰; 刘小宇; 焦炳华

    2013-01-01

    Microbial thermophilic alkaline proteases are proteases with the optimum pH is at alkali range and with high optimum temperature (usually over 60℃),isolated from extreme microorganisms.Thermophilic alkaline proteases have the characteristics of good thermal stability,organic solvent tolerance,denaturant tolerance and long half-life,and have been widely used in many fields such as pharmaceutical industry,thermophilic fermentation,sterilization,as well as in enzymatic synthesis and in waste treatment.This paper briefly introduces the research progress of the enzyme characteristics,the relationship between protein structure and function,and application status and prospect of microbial thermophilic alkaline proteases.%高温碱性蛋白酶是一类最适pH在碱性范围,最适温度在60℃以上的蛋白酶,往往来自于极端环境的微生物中.高温碱性蛋白酶具有热稳定性好、耐有机溶剂、耐变性剂和半寿期长等优良的性质,近年来在医药制剂、高温发酵、化学合成、防杂菌污染、废物处理等领域得到了广泛的应用.本文综述了高温碱性蛋白酶的酶学性质、结构与功能的关系等研究现状及应用前景.

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

    Science.gov (United States)

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

    2016-12-01

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

  14. Production of alkaline protease with immobilized cells of bacillus subtilis PE-11 in various matrices by entrapment technique

    OpenAIRE

    Adinarayana, Kunamneni; Jyothi, Bezawada; Ellaiah, Poluri

    2005-01-01

    The purpose of this investigation was to study the effect ofBacillus subtilis PE-11 cells immobilized in various matrices, such as calcium alginate, k-Carrageenan, ployacrylamide, agar-agar, and gelatin, for the production of alkaline protease. Calcium alginate was found to be an effective and suitable matrix for higher alkaline protease productivity compared to the other matrices studied. All the matrices were selected for repeated batch fermentation. The average specific volumetric producti...

  15. Studies on the Catalytic Properties of Partially Purified Alkaline Proteases from Some Selected Microorganisms

    Directory of Open Access Journals (Sweden)

    Titilayo Olufunke Femi-Ola

    2012-09-01

    Full Text Available Aims: The research was done to study the conditions enhancing catalytic activities of alkaline proteases from Vibro sp., Lactobacillus brevis, Zymomonas sp., Athrobacter sp., Corynebacterium sp. and Bacillus subtilis.Methodology and Results: The proteolytic enzymes were purified in 2-step procedures involving ammonium sulphate precipitation and sephadex G-150 gel permeation chromatography. The upper and lower limits for the specific activities of proteases from the selected microorganisms were estimated at 20.63 and 47.51 units/mg protein with Zymomonas protease having the highest specific activity towards casein as its substrate and purification fold of 3.46, while that ofLactobacillus brevis protease was 8.06. The native molecular weights of these active proteins ranged from 30.4 to 45.7 kDa with Athrobacter sp. protease having the highest weight for its subunits. The proteolytic enzymes had optimum pH range of 8 to 10 and temperature range of 50 to 62 ºC accounting for the percentage relative activity range of 75 to 94% and 71 to 84 % respectively. The activities of Lactobacillus brevis and Bacillus subtilis proteases were maximum at pH 9 and 10 respectively. Lactobacillus brevis protease activity was maximum at temperature of 62 ºC, while beyond this value, a general thermal instability of these active proteins was observed. At above 70 ºC, the catalytic activities of Corynebacterium sp., Vibrio sp., Zymomonas sp. and Arthrobacter sp. proteases were progressively reduced over a period of 120 min of incubation, while Bacillus subtlis and Lactobacillus brevis proteases were relatively stable. Effect of metal ions was investigated on the catalytic activity of protease from the microorganisms. Lactobacillus brevis,Zymomonas sp., Arthrobacter sp., Corynebacterium sp. and Bacillus subtilis protease activities were strongly activated by metal ions such as Ca+2 and Mg+2. Enzyme activities were inhibited strongly by Cu2+ and Hg2+ but were not

  16. Identification of a New Marine Bacterial Strain SD8 and Optimization of Its Culture Conditions for Producing Alkaline Protease.

    Science.gov (United States)

    Cui, Hongxia; Yang, Muyang; Wang, Liping; Xian, Cory J

    2015-01-01

    While much attention has been given to marine microorganisms for production of enzymes, which in general are relatively more stable and active compared to those from plants and animals, studies on alkaline protease production from marine microorganisms have been very limited. In the present study, the alkaline protease producing marine bacterial strain SD8 isolated from sea muds in the Geziwo Qinhuangdao sea area of China was characterized and its optimal culture conditions were investigated. Strain SD8 was initially classified to belong to genus Pseudomonas by morphological, physiological and biochemical characterizations, and then through 16S rDNA sequence it was identified to be likely Pseudomonas hibiscicola. In addition, the culture mediums, carbon sources and culture conditions of strain SD8 were optimized for maximum production of alkaline protease. Optimum enzyme production (236U/mL when cultured bacteria being at 0.75 mg dry weight/mL fermentation broth) was obtained when the isolate at a 3% inoculum size was grown in LB medium at 20 mL medium/100mL Erlenmeyer flask for 48h culture at 30°C with an initial of pH 7.5. This was the first report of strain Pseudomonas hibiscicola secreting alkaline protease, and the data for its optimal cultural conditions for alkaline protease production has laid a foundation for future exploration for the potential use of SD8 strain for alkaline protease production.

  17. Construction and application of recombinant strain for the production of an alkaline protease from Bacillus licheniformis.

    Science.gov (United States)

    Lin, Songyi; Zhang, Meishuo; Liu, Jingbo; Jones, Gregory S

    2015-03-01

    The alkaline protease gene, Apr, from Bacillus licheniformis 2709 was cloned into an expression vector pET - 28b (+), to yield the recombinant plasmid pET-28b (+) - Apr. The pET-28b (+) - Apr was expressed in a high expression strain E. coli BL21. The amino acid sequence deduced from the DNA sequence analysis revealed a 98% identity to that of Bacillus licheniformis 2709. Sodium salt-Polyacrylamide gel electrophoresis (SDS-PAGE) was used to access the protein expression. SDS-PAGE analysis indicated a protein of Mr of 38.8 kDa. The medium components and condition of incubation were optimized for the growth state of a recombinant strain. The optimal composition of production medium was composed of glucose 8 g/L, peptone 8 g/L and salt solution 10 mL. The samples were incubated on a rotary shaker of 180 r/min at 37°C for 24 h.

  18. Cow Dung Substrate for the Potential Production of Alkaline Proteases by Pseudomonas putida Strain AT in Solid-State Fermentation

    OpenAIRE

    Ponnuswamy Vijayaraghavan; Sreekumar Saranya; Samuel Gnana Prakash Vincent

    2014-01-01

    Cow dung and agroresidues were used as the substrates for the production of alkaline proteases by Pseudomonas putida strain AT in solid-state fermentation. Among the various substrates evaluated, cow dung supported maximum (1351±217 U/g) protease production. The optimum conditions for the production of alkaline proteases were a fermentation period of 48 h, 120% (v/w) moisture, pH 9, and the addition of 6% (v/w) inoculum, 1.5% (w/w) trehalose, and 2.0% (w/w) yeast extract to the cow dung subst...

  19. Biophysicochemical characterization of an alkaline protease from Beauveria sp. MTCC 5184 with multiple applications.

    Science.gov (United States)

    Shankar, Shiv; Laxman, Ryali Seeta

    2015-01-01

    This study illustrates the biophysicochemical properties of an alkaline protease, BAP (Beauveria sp. alkaline protease) from Beauveria sp. MTCC 5184. This protease exhibited maximum activity at 50 °C, pH 9.0, and stability in a broad pH range, in the presence of organic solvents, denaturants, as well as detergents. Wash performance studies revealed that BAP was able to remove blood clots/stains from blood-soaked cloth. Peptide mass fingerprinting results demonstrated partial homology of BAP with subtilisin-like proteinase. BAP showed catalytic activity against natural as well as synthetic substrates. Active site characterization of BAP confirmed the involvement of serine, tryptophan, and aspartic acid in catalytic activity. Detailed kinetic and thermodynamic studies of BAP demonstrated that the activation energy (Ea) for casein hydrolysis was 82.55 kJ/M, the specificity constant (Kcat/K m), and the values of ∆G (change in Gibbs free energy) decreased with increase in temperature, whereas ∆H (change in enthalapy) and ∆S (change in entropy) were constant. The results of the present study indicate that BAP has potential for applications as detergent additive, in peptide synthesis, and in basic research.

  20. Functional analysis and structure determination of alkaline protease from Aspergillus flavus.

    Science.gov (United States)

    Syed, Rabbani; Rani, Roja; Sabeena; Masoodi, Tariq Ahmad; Shafi, Gowher; Alharbi, Khalid

    2012-01-01

    Proteases are one of the highest value commercial enzymes as they have broad applications in food, pharmaceutical, detergent, and dairy industries and serve as vital tools in determination of structure of proteins and polypeptides. Multiple application of these enzymes stimulated interest to discover them with novel properties and considerable advancement of basic research into these enzymes. A broad understanding of the active site of the enzyme and of the mechanism of its inactivation is essential for delineating its structure-function relationship. Primary structure analysis of alkaline protease showed 42% of its content to be alpha helix making it stable for three dimensional structure modeling. Homology model of alkaline protease has been constructed using the X-ray structure (3F7O) as a template and swiss model as the workspace. The model was validated by ProSA, SAVES, PROCHECK, PROSAII and RMSD. The results showed the final refined model is reliable. It has 53% amino acid sequence identity with the template, 0.24 Å as RMSD and has -7.53 as Z-score, the Ramachandran plot analysis showed that conformations for 83.4 % of amino acid residues are within the most favored regions and only 0.4% in the disallowed regions.

  1. Purification and characterization of thiol dependent, oxidation-stable serine alkaline protease from thermophilic Bacillus sp.

    Directory of Open Access Journals (Sweden)

    Aysha Kamran

    2015-06-01

    Full Text Available Alkaline serine protease was purified to homogeneity from culture supernatant of a thermophilic, alkaliphilic Bacillus sp. by 80% ammonium sulphate precipitation followed by CM-cellulose and DEAE-cellulose ion exchange column chromatography. The enzyme was purified up to 16.5-fold with 6900 U/mg activity. The protease exhibited maximum activity towards casein at pH 8.0 and at 80 °C. The enzyme was stable at pH 8.0 and 80 °C temperature up to 2 h. The Ca2+ and Mn2+ enhanced the proteolytic activity up to 44% and 36% as compared to control, respectively. However, Zn2+, K+, Ba2+, Co2+, Hg2+ and Cu2+ significantly reduced the enzyme activity. PMSF (phenyl methyl sulphonyl fluoride completely inhibited the protease activity, whereas the activity of protease was stimulated up to two folds in the presence of 5 mM 2-mercaptoethanol. The enzyme was also stable in surfactant (Tween-80 and other commercial detergents (SDS, Triton X-100.

  2. Calcium-induced folding and stabilization of the Pseudomonas aeruginosa alkaline protease.

    Science.gov (United States)

    Zhang, Liang; Conway, James F; Thibodeau, Patrick H

    2012-02-03

    Pseudomonas aeruginosa is an opportunistic pathogen that contributes to the mortality of immunocompromised individuals and patients with cystic fibrosis. Pseudomonas infection presents clinical challenges due to its ability to form biofilms and modulate host-pathogen interactions through the secretion of virulence factors. The calcium-regulated alkaline protease (AP), a member of the repeats in toxin (RTX) family of proteins, is implicated in multiple modes of infection. A series of full-length and truncation mutants were purified for structural and functional studies to evaluate the role of Ca(2+) in AP folding and activation. We find that Ca(2+) binding induces RTX folding, which serves to chaperone the folding of the protease domain. Subsequent association of the RTX domain with an N-terminal α-helix stabilizes AP. These results provide a basis for the Ca(2+)-mediated regulation of AP and suggest mechanisms by which Ca(2+) regulates the RTX family of virulence factors.

  3. Data on optimized production and characterization of alkaline proteases from newly isolated alkaliphiles from Lonar soda lake, India

    Directory of Open Access Journals (Sweden)

    Mukundraj Govindrao Rathod

    2016-09-01

    Full Text Available Alkaline proteases are one of the industrially important enzymes and generally preferred from alkaliphilic sources. Here we have provided the data on optimized production and characterization of alkaline proteases from five newly isolated and identified alkaliphiles from Lonar soda lake, India. The data provided for optimization of physicochemical parameters for maximum alkaline proteases production is based on OVAT (one variable at a time approach. Alkaline protease production (U/mL recorded by using different agro industrial residues is included in the given data. Further readers can find more information in our previously published research article where we have already described about the methods used and comparative analysis of the data recorded regarding optimized production, characterization and application of alkaline proteases isolated from Lonar soda lake isolates (http://dx.doi.org/10.1016/j.bcab.2016.06.002 [1]. The data provided here by us is useful to other researchers for setting up various suitable statistical models to perform optimization studies other than OVAT approach.

  4. The selection of alkaline protease-producing yeasts from marine environments and evaluation of their bioactive peptide production

    Institute of Scientific and Technical Information of China (English)

    LI Jing; CHI Zhenming; WANG Xianghong; PENG Ying; CHI Zhe

    2009-01-01

    A total of 400 yeast strains from seawater, sediments, saltern mud, marine fish guts, and marine algae were obtained. The protease activity of the yeast cultures was estimated, after which four strains (HN3.11, N11b, YF04C and HN4.9) capable of secreting extracellular alkaline protease were isolated. The isolated strains were identified as Aureobasidium pullulans, Yarrowia lipolytica, Issatchenkia orientalis and Cryptococcus cf. aureus. The optimal pH of the protease activity produced by strains HN3.11, YF04C, and HN4.9 was 9.0, while that of the protease produced by strain N11b was 10.0. The optimal temperature for protease activity was 45°C for strains HN3.11, N11b, and YF04C, and 50°C for strain HN4.9. After digestion of shrimp (Penaeus vannamei) protein and spirulina (Arthospira platensis) protein with the four crude alkaline proteases, the filtrate from spirulina (Arthrospira platensis) powder digested by the crude alkaline protease of strain HN3.11 was found to have the highest antioxidant activity (61.4%) and the highest angiotensin I converting enzyme (ACE)-inhibitory activities (68.4%). The other filtrates had much lower antioxidant activity and ACE-inhibitory activities.

  5. Statistical Approach for Optimization of Physiochemical Requirements on Alkaline Protease Production from Bacillus licheniformis NCIM 2042

    Directory of Open Access Journals (Sweden)

    Biswanath Bhunia

    2012-01-01

    Full Text Available The optimization of physiochemical parameters for alkaline protease production using Bacillus licheniformis NCIM 2042 were carried out by Plackett-Burman design and response surface methodology (RSM. The model was validated experimentally and the maximum protease production was found 315.28 U using optimum culture conditions. The protease was purified using ammonium sulphate (60% precipitation technique. The HPLC analysis of dialyzed sample showed that the retention time is 1.84 min with 73.5% purity. This enzyme retained more than 92% of its initial activity after preincubation for 30 min at 37∘C in the presence of 25% v/v DMSO, methanol, ethanol, ACN, 2-propanol, benzene, toluene, and hexane. In addition, partially purified enzyme showed remarkable stability for 60 min at room temperature, in the presence of anionic detergent (Tween-80 and Triton X-100, surfactant (SDS, bleaching agent (sodium perborate and hydrogen peroxide, and anti-redeposition agents (Na2CMC, Na2CO3. Purified enzyme containing 10% w/v PEG 4000 showed better thermal, surfactant, and local detergent stability.

  6. Virtual screening of novel reversible inhibitors for marine alkaline protease MP.

    Science.gov (United States)

    Ji, Xiaofeng; Zheng, Yuan; Wang, Wei; Sheng, Jun; Hao, Jianhua; Sun, Mi

    2013-11-01

    Marine alkaline protease (MP,(2) accession no. ACY25898) is produced by a marine bacterium strain isolated from Yellow Sea sediment in China. Previous research has shown that this protease is a cold-adapted enzyme with antioxidant activity that could be used as a detergent additive. Owing to its instability in the liquid state, MP's application in liquid detergents was limited. Therefore, the discovery of reversible MP inhibitors to stabilize the protease was imperative. Here, we used the X-ray structure of MP and recompiled AutoDock 4.2 with refined Zn(2+) characters to screen the free chemical database ZINC. After completing the docking procedure, we applied strategies including the "initial filter", consensus scoring and pharmocophore model to accelerate the process and improve the virtual screening success rate. The "initial filter" was built based on the docking results of boronic acid derivatives validated as reversible inhibitors of MP by our previous studies. Finally, ten compounds were purchased or synthetized to test their binding affinity for MP. Three of the compounds could reversibly inhibit MP with apparent Ki values of 0.8-1.2 mmol. These active compounds and their binding modes provide useful information for understanding the molecular mechanism of reversible MP inhibition. The results may also serve as the foundation for further screening and design of reversible MP inhibitors.

  7. Selection of Suitable Carbon, Nitrogen and Sulphate Source for the Production of Alkaline Protease by Bacillus licheniformis NCIM-2042

    Directory of Open Access Journals (Sweden)

    Biswanath BHUNIA

    2010-06-01

    Full Text Available In this study, selection of suitable carbon, nitrogen and sulphate sources were carried out by one-variable-at-time approach for the production of alkaline protease enzyme by Bacillus licheniformis NCIM-2042. Maximum levels of alkaline protease were found in culture media supplemented with magnesium sulphate, starch and soybean meal as a good sulphate, carbon and nitrogen sources which influenced the maximum yield of this enzyme (137.694.57, 135.231.73 and 134.741.77, respectively in comparison with the other sulphate, carbon and nitrogen sources.

  8. Selection of Suitable Carbon, Nitrogen and Sulphate Source for the Production of Alkaline Protease by Bacillus licheniformis NCIM-2042

    Directory of Open Access Journals (Sweden)

    Biswanath BHUNIA

    2010-06-01

    Full Text Available In this study, selection of suitable carbon, nitrogen and sulphate sources were carried out by one-variable-at-time approach for the production of alkaline protease enzyme by Bacillus licheniformis NCIM-2042. Maximum levels of alkaline protease were found in culture media supplemented with magnesium sulphate, starch and soybean meal as a good sulphate, carbon and nitrogen sources which influenced the maximum yield of this enzyme (137.69�4.57, 135.23�1.73 and 134.74�1.77, respectively in comparison with the other sulphate, carbon and nitrogen sources.

  9. Purification and biochemical characterization of a novel alkaline protease produced by Penicillium nalgiovense.

    Science.gov (United States)

    Papagianni, M; Sergelidis, D

    2014-04-01

    Penicillium nalgiovense PNA9 produces an extracellular protease during fermentation with characteristics of growth-associated product. Enzyme purification involved ammonium sulfate precipitation, dialysis, and ultrafiltration, resulting in 12.1-fold increase of specific activity (19.5 U/mg). The protein was isolated through a series of BN-PAGE and native PAGE runs. ESI-MS analysis confirmed the molecular mass of 45.2 kDa. N-Terminal sequencing (MGFLKLLKGSLATLAVVNAGKLLTANDGDE) revealed 93 % similarity to a Penicillium chrysogenum protease, identified as major allergen. The protease exhibits simple Michaelis-Menten kinetics and K m (1.152 mg/ml), V max (0.827 mg/ml/min), and k cat (3.2 × 10(2)) (1/s) values against azocasein show that it possesses high substrate affinity and catalytic efficiency. The protease is active within 10-45 °C, pH 4.0-10.0, and 0-3 M NaCl, while maximum activity was observed at 35 °C, pH 8.0, and 0.25 M NaCl. It is active against the muscle proteins actin and myosin and inactive against myoglobin. It is highly stable in the presence of non-ionic surfactants, hydrogen peroxide, BTNB, and EDTA. Activity was inhibited by SDS, Mn(2+) and Zn(2+), and by the serine protease inhibitor PMSF, indicating the serine protease nature of the enzyme. These properties make the novel protease a suitable candidate enzyme in meat ripening and other biotechnological applications.

  10. Selective modulation of the CD4 molecular complex by Pseudomonas aeruginosa alkaline protease and elastase

    DEFF Research Database (Denmark)

    Pedersen, B K; Kharazmi, A; Theander, T G

    1987-01-01

    The binding of monoclonal antibodies against CD4 was specifically inhibited by treatment of human CD4+ cells with either alkaline protease (AP) or elastase (Ela), purified from Pseudomonas aeruginosa. Binding of antibodies against CD3 (pan T), CD5 (pan T), CD8 (T suppressor/cytotoxic), HLA-ABC, HLA......-DR, HLA-DQ, HLA-DP/DR, and beta 2 microglobulin was not inhibited by AP or Ela. Heat-inactivation of the proteases at 65 degrees C for 20 min or treatment with the metal chelator EDTA abolished the inhibitory activity of both proteases. These findings may serve to develop novel immunological methods...

  11. Secretory expression of thermostable alkaline protease from Bacillus stearothermophilus FI by using native signal peptide and α-factor secretion signal in Pichia pastoris.

    Science.gov (United States)

    Latiffi, Amaliawati Ahmad; Salleh, Abu Bakar; Rahman, Raja Noor Zaliha Raja Abd; Oslan, Siti Nurbaya; Basri, Mahiran

    2013-01-01

    The thermostable alkaline protease from Bacillus stearothermophilus F1 has high potential for industrial applications, and attempt to produce the enzyme in yeast for higher yield was undertaken. Secretory expression of F1 protease through yeast system could improve enzyme's capability, thus simplifying the purification steps. Mature and full genes of F1 protease were cloned into Pichia pastoris expression vectors (pGAPZαB and pPICZαB) and transformed into P. pastoris strains (GS115 and SMD1168H) via electroporation method. Recombinant F1 protease under regulation constitutive GAP promoter revealed that the highest expression was achieved after 72 h cultivation. While inducible AOX promoter showed that 0.5% (v/v) methanol was the best to induce expression. It was proven that constitutive expression strategy was better than inducible system. The α-secretion signal from the plasmid demonstrated higher secretory expression level of F1 protease as compared to native Open Reading Frame (ORF) in GS115 strain (GE6GS). Production medium YPTD was found to be the best for F1 protease expression with the highest yield of 4.13 U/mL. The protein was expressed as His-tagged fusion protein with a size about 34 kDa.

  12. Purification and characterization of alkaline-thermostable protease enzyme from Pitaya (Hylocereus polyrhizus) waste: a potential low cost of the enzyme.

    Science.gov (United States)

    Amid, Mehrnoush; Manap, Mohd Yazid A B D; Zohdi, Nor Khanani

    2014-01-01

    The thermoalkaline protease enzyme from pitaya (Hylocereus polyrhizus) waste was purified by a factor of 221.2 with 71.3% recovery using ammonium sulphate precipitation, gel filtration, and cation exchange chromatography. Gel filtration chromatography together with sodium dodecyl sulphate gel electrophoresis (SDS-PAGE) revealed that the enzyme is monomeric with a molecular weight of 26.7 kDa. The apparent K m and V max of the protease were 2.8 mg/mL and 31.20 u/min, respectively. The optimum pH and temperature were 8.0 and 70°C. The enzyme was highly active and stable over a wide pH range (from pH 3.0 to pH 11.0 with the optimum activity at pH 8.0). The protease has broad specificity toward azocasein, casein, hemoglobin, and gelatine. Activity of the enzyme was inhibited by Fe(2+) and Zn(2+), while protease activity was increased in the presence of Ca(2+) and Mg(2+) and Cu(2+) by factors of 125%, 110%, and 105%, respectively. The alkaline protease showed extreme stability toward surfactants and oxidizing agent. The purified protease exhibited extreme stability in the presence of organic solvents and inhibitors. In addition, the enzyme was relativity stable toward organic solvents and chelating agents, such as ethylenediaminetetraacetic acid (EDTA). The enzyme, derived from pitaya peel, possesses unique characteristics and could be used in various industrial and biotechnological applications.

  13. Growth energetics of an alkaline serine protease-producing strain of Bacillus clausii during continuous cultivation

    DEFF Research Database (Denmark)

    Christiansen, Torben; Nielsen, Jens

    2002-01-01

    the theoretical co-factor and building block requirements needed for biomass formation were calculated. Using the stoichiometric models and data for growth on glucose and citrate the amount of ATP needed for biomass synthesis was estimated to 42.0 mmol ATP/gDW, the P/O ratio to 0.68 and the ATP maintenance to 2......Glucose-limited chemostats were used to determine the growth yields of biomass of Bacillus clausii PP 473-8 producing an alkaline serine protease Savinase (Novozymes A/S, Bagsvaerd, Denmark) and a low yield of biomass on oxygen was observed. The energy metabolism was investigated further by setting...... up simple stoichiometric models for growth on glucose and citrate. In order to determine the parameters in the models, a macromolecular biomass composition was determined based on measured values of protein and RNA combined with literature data. From the macromolecular composition of the biomass...

  14. 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.

  15. Purification and characterization of an alkaline protease from Bacillus licheniformis UV-9 for detergent formulations

    Directory of Open Access Journals (Sweden)

    Muhammad Nadeem

    2013-04-01

    Full Text Available Alkaline protease produced by mutant strain B. licheniformis UV-9 was purified and characterized for its exploitationin detergent formulation. The enzyme was purified to homogeneity by employing ammonium sulphate precipitation andsephadex G-100 gel filtration chromatography with a 36.83 fold increase in specific activity and 11% recovery. The molecularweight of the protease was found to be 36.12 kDa by SDS-PAGE. The Km and Vmax values exhibited by purified proteasewere 5 mg/ml and 61.58ìM/ml/min, respectively, using casein as substrate. The enzyme exhibited highest activity at pH 11 andtemperature 60°C. Stability studies showed that the enzyme retained higher than 80% residual activity in the pH and temperature ranges of 8 to 11 and 30 to 50°C, respectively. However, in the presence of 10 mM Ca2+ ions the enzyme tained morethan 90% of its residual activity at pH 11 and temperature 60°C. Phenyl methyl sulphonyl fluoride (PMSF completelyinhibited the enzyme activity suggesting that it was serine protease. Among metal ions, the Mg2+ and Ca2+ ions enhancedactivity up to 128% and 145%, respectively. The purified enzyme showed extreme stability towards various surfactantssuch as Tween-20, Tween- 45, Tween-65 and Triton X-45. In addition, the enzyme also exhibited more than 100% residualactivity in the presence of oxidizing agents, H2O2 and sodium perborate. These biochemical properties indicate the potentialuse of B. licheniformis UV-9 enzyme in laundry detergents.

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

    Science.gov (United States)

    Huang, Rong; Yang, Qingjun; Feng, Hong

    2015-02-01

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

  17. Simultaneous production of alkaline lipase and protease by antibiotic and heavy metal tolerant Pseudomonas aeruginosa.

    Science.gov (United States)

    Bisht, Deepali; Yadav, Santosh Kumar; Gautam, Pallavi; Darmwal, Nandan Singh

    2013-09-01

    An efficient bacterial strain capable of simultaneous production of lipase and protease in a single production medium was isolated. Thirty six bacterial strains, isolated from diverse habitats, were screened for their lipolytic and proteolytic activity. Of these, only one bacterial strain was found to be lipase and protease producer. The 16S rDNA sequencing and phylogenetic analyses revealed that strain (NSD-09) was in close identity to Pseudomonas aeruginosa. The maximum lipase (221.4 U/ml) and protease (187.9 U/ml) activities were obtained after 28 and 24 h of incubation, respectively at pH 9.0 and 37 °C. Castor oil and wheat bran were found to be the best substrate for lipase and protease production, respectively. The strain also exhibited high tolerance to lead (1450 µg/ml) and chromium (1000 µg/ml) in agar plates. It also showed tolerance to other heavy metals, such as Co(+2) , Zn(+2) , Hg(+2) , Ni(+2) and Cd(+2) . Therefore, this strain has scope for tailing bioremediation. Presumably, this is the first attempt on P. aeruginosa to explore its potential for both industrial and environmental applications.

  18. An Alkaline Protease from Bacillus pumilus MP 27: Functional Analysis of its Binding Model towards its Applications as Detergent Additive

    Directory of Open Access Journals (Sweden)

    Mehak Baweja

    2016-08-01

    Full Text Available A proteolytic strain of Bacillus pumilus MP 27 was isolated from water samples of Southern ocean produced alkaline protease. Since protease production need expensive ingredients, an economically viable process was developed by using low cost carbon source, wheat straw, supplemented with peptone. This protease was active within temperature ranges 10˚C -70˚C at pH 9. This process was optimized by response surface methodology using a Box Bekhman design by Design Expert 7.0 software that increased the protease activity to 776.5 U/ml. Moreover, the enzyme was extremely stable at a broad range of temperature and pH retaining 69% of its activity at 50 ºC and 70% at pH 11. The enzyme exhibited excellent compatibility with surfactants and commercial detergents, showing 87% stability with triton X-100 and ̴ 100% stability with Tide commercial detergent. The results of the wash performance analysis demonstrated considerably good de-staining at 50ºC and 4ºC with low supplementation (109 U/ml. Molecular modeling of the protease revealed the presence of serine proteases, subtilase family and serine active site and further docking supported the association of catalytic site with the various substrates. Certainly, such protease can be considered as a good detergent additive in detergent industry with a possibility to remove the stains effectively even in a cold wash.

  19. An Alkaline Protease from Bacillus pumilus MP 27: Functional Analysis of Its Binding Model toward Its Applications As Detergent Additive.

    Science.gov (United States)

    Baweja, Mehak; Tiwari, Rameshwar; Singh, Puneet K; Nain, Lata; Shukla, Pratyoosh

    2016-01-01

    A proteolytic strain of Bacillus pumilus MP 27 was isolated from water samples of Southern ocean produced alkaline protease. Since protease production need expensive ingredients, an economically viable process was developed by using low cost carbon source, wheat straw, supplemented with peptone. This protease was active within temperature ranges 10-70°C at pH 9. This process was optimized by response surface methodology using a Box Bekhman design by Design Expert 7.0 software that increased the protease activity to 776.5 U/ml. Moreover, the enzyme was extremely stable at a broad range of temperature and pH retaining 69% of its activity at 50°C and 70% at pH 11. The enzyme exhibited excellent compatibility with surfactants and commercial detergents, showing 87% stability with triton X-100 and 100% stability with Tide commercial detergent. The results of the wash performance analysis demonstrated considerably good de-staining at 50 and 4°C with low supplementation (109 U/ml). Molecular modeling of the protease revealed the presence of serine proteases, subtilase family and serine active site and further docking supported the association of catalytic site with the various substrates. Certainly, such protease can be considered as a good detergent additive in detergent industry with a possibility to remove the stains effectively even in a cold wash.

  20. 包衣微丸型碱性蛋白酶的制备%Preparation of coated granule alkaline protease

    Institute of Scientific and Technical Information of China (English)

    马俊孝; 张赞剑; 史衍鲁; 卢彦梅; 刘伟

    2012-01-01

    开发了一种包衣微丸型碱性蛋白酶的制备工艺,结果表明:30 L发酵中罐发酵50 h比酶活可达4.26×104 U/mL,发酵液经絮凝处理、板框压滤、膜浓缩后可制成酶活达300 000 U/mL的酶浓缩液.经流化后制得含酶颗粒,再包裹薄层后,得包衣微丸型碱性蛋白酶.对制备的包衣微丸型碱性蛋白酶的稳定性、去污效果等指标进行了评估.制备的包衣微丸型碱性蛋白酶产品在严苛条件下的稳定性与国外产品( Savinase 8.0T、PuraFast 2000HS)相当,产品暴露在37℃、75%湿度下8周后仍可保持74%的酶活力,产品的去污效果优于国外产品.制备的包衣微丸型碱性蛋白酶颗粒大小均匀,流动性和分散性好,对外界高温、高湿等不良环境具有很强的抵抗能力,适于工业化生产.%A process was developed for the preparing of coated granule alkaline protease. The method for preparing enzyme solution included fermentation, flocculation sedimentation, filtration, and ultrafiltration by membrane. The final products were prepared by producing a core containing enzymes and spraying the coating layer onto the core in a fluid-bed. The stability and detergency of the coated granule were investigated by accelerated experiments, and the characteristics of products were evaluated. The main results were as follows; the activity of protease was about 4. 26 x 10 U/mL after cultivating for 50 h in 30 L fer-mentor; The granule alkaline protease was characterized by a uniform particle size distribution, a high rate of finished products, and a free-flowing granulate (300 000 U/mL). When exposed at 37 ? and 75% relative humidity for 8 weeks, the coated granule retained 74% of the original activity. Furthermore , the detergency was better than that of a commercial product.

  1. Alkaline serine protease AprE plays an essential role in poly-γ-glutamate production during natto fermentation.

    Science.gov (United States)

    Kada, Shigeki; Ishikawa, Atsushi; Ohshima, Yoshifumi; Yoshida, Ken-ichi

    2013-01-01

    Natto is a traditional Japanese food made from soybeans fermented by natto starter strains of Bacillus subtilis natto. It has been suggested that extracellular protease activity released by the bacteria are involved in the production of poly-γ-glutamate (γ-PGA) during natto fermentation. One of the natto starters, strain r22, possesses at least seven genes, each of which encoded an extracellular protease orthologous to its counterpart in B. subtilis 168, aprE, bpr, epr, mpr, nprE, vpr, and wprA, but it was found to lack nprB. Inactivating the aprE ortholog alone resulted in a severe decrease in γ-PGA production and in the total extracellular protease activity. The defect in γ-PGA production of the mutant lacking the aprE ortholog was complemented when the medium was supplemented with sufficient glutamate. These results suggest that the alkaline serine protease encoded by aprE plays an indispensable role in supplying materials to produce γ-PGA. On the other hand, simultaneous inactivation of all the protease genes except for aprE did not significantly affect either γ-PGA production or total protease activity.

  2. Production of extracellular alkaline protease by new halotolerant alkaliphilic Bacillus sp. NPST-AK15 isolated from hyper saline soda lakes

    Directory of Open Access Journals (Sweden)

    Abdelnasser S.S. Ibrahim

    2015-05-01

    Conclusions: A new halotolerant alkaliphilic alkaline protease producing Bacillus sp. NPST-AK15 was isolated from soda lakes. Optimization of various fermentation parameters resulted in an increase of enzyme yield by 22.8 fold, indicating the significance of optimization of the fermentation parameters to obtain commercial yield of the enzyme. NPST-AK15 and its extracellular alkaline protease with salt tolerance signify their potential applicability in the laundry industry and other applications.

  3. Purification and Characterization of Alkaline-Thermostable Protease Enzyme from Pitaya (Hylocereus polyrhizus Waste: A Potential Low Cost of the Enzyme

    Directory of Open Access Journals (Sweden)

    Mehrnoush Amid

    2014-01-01

    Full Text Available The thermoalkaline protease enzyme from pitaya (Hylocereus polyrhizus waste was purified by a factor of 221.2 with 71.3% recovery using ammonium sulphate precipitation, gel filtration, and cation exchange chromatography. Gel filtration chromatography together with sodium dodecyl sulphate gel electrophoresis (SDS-PAGE revealed that the enzyme is monomeric with a molecular weight of 26.7 kDa. The apparent Km and Vmax of the protease were 2.8 mg/mL and 31.20 u/min, respectively. The optimum pH and temperature were 8.0 and 70°C. The enzyme was highly active and stable over a wide pH range (from pH 3.0 to pH 11.0 with the optimum activity at pH 8.0. The protease has broad specificity toward azocasein, casein, hemoglobin, and gelatine. Activity of the enzyme was inhibited by Fe2+ and Zn2+, while protease activity was increased in the presence of Ca2+ and Mg2+ and Cu2+ by factors of 125%, 110%, and 105%, respectively. The alkaline protease showed extreme stability toward surfactants and oxidizing agent. The purified protease exhibited extreme stability in the presence of organic solvents and inhibitors. In addition, the enzyme was relativity stable toward organic solvents and chelating agents, such as ethylenediaminetetraacetic acid (EDTA. The enzyme, derived from pitaya peel, possesses unique characteristics and could be used in various industrial and biotechnological applications.

  4. Enhancement of Alkaline Protease Activity and Stability via Covalent Immobilization onto Hollow Core-Mesoporous Shell Silica Nanospheres

    Directory of Open Access Journals (Sweden)

    Abdelnasser Salah Shebl Ibrahim

    2016-01-01

    Full Text Available The stability and reusability of soluble enzymes are of major concerns, which limit their industrial applications. Herein, alkaline protease from Bacillus sp. NPST-AK15 was immobilized onto hollow core-mesoporous shell silica (HCMSS nanospheres. Subsequently, the properties of immobilized proteases were evaluated. Non-, ethane- and amino-functionalized HCMSS nanospheres were synthesized and characterized. NPST-AK15 was immobilized onto the synthesized nano-supports by physical and covalent immobilization approaches. However, protease immobilization by covalent attachment onto the activated HCMSS–NH2 nanospheres showed highest immobilization yield (75.6% and loading capacity (88.1 μg protein/mg carrier and was applied in the further studies. In comparison to free enzyme, the covalently immobilized protease exhibited a slight shift in the optimal pH from 10.5 to 11.0, respectively. The optimum temperature for catalytic activity of both free and immobilized enzyme was seen at 60 °C. However, while the free enzyme was completely inactivated when treated at 60 °C for 1 h the immobilized enzyme still retained 63.6% of its initial activity. The immobilized protease showed higher Vmax, kcat and kcat/Km, than soluble enzyme by 1.6-, 1.6- and 2.4-fold, respectively. In addition, the immobilized protease affinity to the substrate increased by about 1.5-fold. Furthermore, the enzyme stability in various organic solvents was significantly enhanced upon immobilization. Interestingly, the immobilized enzyme exhibited much higher stability in several commercial detergents including OMO, Tide, Ariel, Bonux and Xra by up to 5.2-fold. Finally, the immobilized protease maintained significant catalytic efficiency for twelve consecutive reaction cycles. These results suggest the effectiveness of the developed nanobiocatalyst as a candidate for detergent formulation and peptide synthesis in non-aqueous media.

  5. Enhancement of Alkaline Protease Activity and Stability via Covalent Immobilization onto Hollow Core-Mesoporous Shell Silica Nanospheres.

    Science.gov (United States)

    Ibrahim, Abdelnasser Salah Shebl; Al-Salamah, Ali A; El-Toni, Ahmed M; Almaary, Khalid S; El-Tayeb, Mohamed A; Elbadawi, Yahya B; Antranikian, Garabed

    2016-01-29

    The stability and reusability of soluble enzymes are of major concerns, which limit their industrial applications. Herein, alkaline protease from Bacillus sp. NPST-AK15 was immobilized onto hollow core-mesoporous shell silica (HCMSS) nanospheres. Subsequently, the properties of immobilized proteases were evaluated. Non-, ethane- and amino-functionalized HCMSS nanospheres were synthesized and characterized. NPST-AK15 was immobilized onto the synthesized nano-supports by physical and covalent immobilization approaches. However, protease immobilization by covalent attachment onto the activated HCMSS-NH₂ nanospheres showed highest immobilization yield (75.6%) and loading capacity (88.1 μg protein/mg carrier) and was applied in the further studies. In comparison to free enzyme, the covalently immobilized protease exhibited a slight shift in the optimal pH from 10.5 to 11.0, respectively. The optimum temperature for catalytic activity of both free and immobilized enzyme was seen at 60 °C. However, while the free enzyme was completely inactivated when treated at 60 °C for 1 h the immobilized enzyme still retained 63.6% of its initial activity. The immobilized protease showed higher V(max), k(cat) and k(cat)/K(m), than soluble enzyme by 1.6-, 1.6- and 2.4-fold, respectively. In addition, the immobilized protease affinity to the substrate increased by about 1.5-fold. Furthermore, the enzyme stability in various organic solvents was significantly enhanced upon immobilization. Interestingly, the immobilized enzyme exhibited much higher stability in several commercial detergents including OMO, Tide, Ariel, Bonux and Xra by up to 5.2-fold. Finally, the immobilized protease maintained significant catalytic efficiency for twelve consecutive reaction cycles. These results suggest the effectiveness of the developed nanobiocatalyst as a candidate for detergent formulation and peptide synthesis in non-aqueous media.

  6. ISOLASI DAN KARAKTERISASI PROTEASE ALKALIN DARI ISOLAT BAKTERI LIMBAH TERNAK DI EXFARM FAKULTAS PETERNAKAN UNSOED

    Directory of Open Access Journals (Sweden)

    Zusfahair

    2011-05-01

    Full Text Available Protease is one of the widely used enzymes for the industry. The potential resource of microorganism that produced protease is milk cow waste. In this research, isolation and characterization has been done toward isolated protease from milk cow waste of the Exfarm’s Animal Husbandry Faculty at University of Jenderal Soedirman, Purwokerto. The research used experiment method and the parameters observed were the genus of bacteria which produce protease and the activity of protease. The characterizations of protease were determination of optimum pH and temperature, the influence of metal ions, EDTA, surfactant, and commercial detergent toward enzyme activity, and also the study of enzyme stability. The results from the research showed that the isolated bacteria from the Exfarm’s of Animal Husbandry Faculty of UNSOED, which produced protease was Salmonella sp. Characterization of isolated Salmonella sp. from 45% ammonium sulphate fraction indicated that the optimum temperature was 50 ºC, optimum pH was 8, the enzyme was activated by Ca2+ dan Mg2+ ion, whereas it was inhibited by Zn2+, Cu2+ ions and EDTA. The addition of Tween-80 with the concentration of 0.2% and 0.4% increased protease activity, however the addition of Tween-80 with concentration higher than 0.6% decreased the protease activity. Enzyme protease from isolated Salmonella sp. was relatively stable with the addition of commercial detergent such as Attack, Surf, and Bukrim.

  7. Biochemical analysis and investigation on the prospective applications of alkaline protease from a Bacillus cereus strain.

    Science.gov (United States)

    Saleem, Mahjabeen; Rehman, Atiqa; Yasmin, Riffat; Munir, Bushra

    2012-06-01

    Proteases have prospective financial and environment-friendly applications; hence attention is focused currently on the finding of new protease producing microorganism so as to meet the requirements of industry. A thermophilic bacterial strain producing extracellular protease activity was isolated from soil and identified as Bacillus cereus by analysis of 16S rRNA. Protease production by the microorganism was improved by studying the impact of the type of nitrogen and carbon source, fermentation period, growth temperature and initial pH of the culture medium in cultivation optimization experiments. The enzyme was purified to homogeneity in two step procedure involving Sephadex G-75 and Q-Sepharose chromatography. The molecular weight of purified enzyme was found to be 58 kDa by SDS-PAGE. Protease exhibited a pH and temperature optima of 7.5 and 60°, respectively. The enzyme was active in the pH range of 6.0-9.0 and stable up to 70°C. Histological analysis of protease treated goat and cow skin pelts showed complete removal of non leather forming structures such as hair shaft, hair follicles and glandular structures. The protease showed the stain removing property from blood stained cotton cloth and found to be compatible with six commercially available detergents. The protease could release peptides from natural proteins after digestion of coagulated egg albumin and blood clot.

  8. Studies of alkaline protease with plasmin activity in milk%牛乳中具纤溶活性碱性蛋白酶的研究

    Institute of Scientific and Technical Information of China (English)

    白英; 陈振涛

    2012-01-01

    One alkaline protease with plasmin activity was isolated from late-lactation bovine milk. The enzyme was purified to electrophoret-ic homogeneity using ε—Amino caproic acid, CM Sepharose Fast Flow ion exchange chromatography, Lysine—Sepharose affinity chromatogra-phy. Purify multiple is 2.11. The isoeleetrie point of protease was 7.8. The molecular weight was 47.0 ku measured by SDS—PAGE. Activity of protease was completely inhibited by the serine protease inhibitor, PMSF, indicated that it belongs to the serine protease family. The enzyme had a high degrading activity for casein.%通过ε-氨基己酸的解离柞用,利用CM Sepharose Fast Flow凝胶过滤层析、Lysine-Sepharose(赖氨酸-琼脂糖凝胶)亲和层析的方法,通过SDS -聚丙烯酰胺凝胶电泳纯度鉴定,从末乳中分离出一种具有纤溶活性的碱性蛋白酶.采用凝胶色谱和等电聚焦法对其分子量和等电点进行测定.结果表明:该蛋白酶为单体蛋白,纯化倍数2.11,分子量为47.0 ku,等电点7.8.纯化后酶的活性可完全被PMSF抑制剂抑制,属于丝氨酸蛋白酶家族.对酪蛋白具有一定的水解能力.

  9. High temperature and pressure alkaline electrolysis

    DEFF Research Database (Denmark)

    Allebrod, Frank; Chatzichristodoulou, Christodoulos; Mogensen, Mogens Bjerg

    2013-01-01

    the operational temperature and pressure to produce pressurized hydrogen at high rate (m3 H2·h-1·m-2 cell area) and high electrical efficiency. This work describes an exploratory technical study of the possibility to produce hydrogen and oxygen with a new type of alkaline electrolysis cell at high temperatures...... for immobilization of aqueous KOH solutions. Electrolysis cells with this electrolyte and metal foam based gas diffusion electrodes were successfully demonstrated at temperatures up to 250 °C at 40 bar. Different electro-catalysts were tested in order to reduce the oxygen and hydrogen overpotentials. Current...

  10. Optimization and characterization of alkaline protease and carboxymethyl-cellulase produced by Bacillus pumillus grown on Ficus nitida wastes

    Directory of Open Access Journals (Sweden)

    Eman Zakaria Gomaa

    2013-01-01

    Full Text Available The potentiality of 23 bacterial isolates to produce alkaline protease and carboxymethyl-cellulase (CMCase on Ficus nitida wastes was investigated. Bacillus pumillus ATCC7061 was selected as the most potent bacterial strain for the production of both enzymes. It was found that the optimum production of protease and CMCase were recorded at 30 °C, 5% Ficus nitida leaves and incubation period of 72 h. The best nitrogen sources for protease and CMCase production were yeast extract and casein, respectively. Also maximum protease and CMCase production were reported at pH 9 and pH 10, respectively. The enzymes possessed a good stability over a pH range of 8-10, expressed their maximum activities at pH10 and temperature range of 30-50 °C, expressed their maximum activities at 50 °C. Ions of Hg2+, Fe2+ and Ag+ showed a stimulatory effect on protease activity and ions of Fe2+, Mg2+, Ca2+, Cu2+ and Ag+ caused enhancement of CMCase activity. The enzymes were stable not only towards the nonionic surfactants like Triton X-100 and Tween 80 but also the strong anionic surfactant, SDS. Moreover, the enzymes were not significantly inhibited by EDTA or cystein. Concerning biotechnological applications, the enzymes retained (51-97% of their initial activities upon incubation in the presence of commercials detergents for 1 h. The potential use of the produced enzymes in the degradation of human hair and cotton fabric samples were also assessed.

  11. Optimization and characterization of alkaline protease and carboxymethyl-cellulase produced by Bacillus pumillus grown on Ficus nitida wastes.

    Science.gov (United States)

    Gomaa, Eman Zakaria

    2013-01-01

    The potentiality of 23 bacterial isolates to produce alkaline protease and carboxymethyl-cellulase (CMCase) on Ficus nitida wastes was investigated. Bacillus pumillus ATCC7061 was selected as the most potent bacterial strain for the production of both enzymes. It was found that the optimum production of protease and CMCase were recorded at 30 °C, 5% Ficus nitida leaves and incubation period of 72 h. The best nitrogen sources for protease and CMCase production were yeast extract and casein, respectively. Also maximum protease and CMCase production were reported at pH 9 and pH 10, respectively. The enzymes possessed a good stability over a pH range of 8-10, expressed their maximum activities at pH10 and temperature range of 30-50 °C, expressed their maximum activities at 50 °C. Ions of Hg(2+), Fe2+ and Ag(+) showed a stimulatory effect on protease activity and ions of Fe(2+), Mg(2+), Ca(2+), Cu(2+) and Ag(+) caused enhancement of CMCase activity. The enzymes were stable not only towards the nonionic surfactants like Triton X-100 and Tween 80 but also the strong anionic surfactant, SDS. Moreover, the enzymes were not significantly inhibited by EDTA or cystein. Concerning biotechnological applications, the enzymes retained (51-97%) of their initial activities upon incubation in the presence of commercials detergents for 1 h. The potential use of the produced enzymes in the degradation of human hair and cotton fabric samples were also assessed.

  12. Enhanced Productivity of Serine Alkaline Protease by Bacillus sp. Using Soybean as Substrate

    Directory of Open Access Journals (Sweden)

    Saurabh, S.

    2007-01-01

    Full Text Available The growth and protease production by Bacillus sp. (SBP-29 was examined for poultry processing industries. The maximum protease activity was 3028 U/mL using 1.5% (w/v of soybean meal as substrate. Soybean meal is an inexpensive and readily available, thus it can be used as the cost effective crude material for the production of an extracellular protease. Inorganic nitrogen sources proved to be less favorable, for protease production as strong catabolic repression was observed with ammonium ions. A maximum of 3208 U/mL of protease was produced in 18 h in a 10L bioreactor. The enzyme has temperature and pH optima of 60°C and 9.5 respectively. However, the temperature stability range is from 20-90 °C and pH stability range is from 6.0–12.0. The protease was completely inhibited by phenylmethylsulfonyl fluoride (PMSF and diodopropyl fluorophosphate (DFP, with little increase (10-15% in the production of upon addition of Ca++ and Mg++.

  13. High Temperature and Pressure Alkaline Electrolysis

    DEFF Research Database (Denmark)

    Allebrod, Frank

    and oxygen with a new type of alkaline electrolysis cell at high temperatures and pressures. To perform measurements under high pressure and at elevated temperatures it was necessary to build a measurement system around an autoclave which could stand high temperatures up to 250 °C and pressures up to 200 bar...... as well as extremely caustic environments. Based on a literature study to identify resistant materials for these conditions, Inconel 600 was selected among the metals which are available for autoclave construction. An initial single atmosphere high temperature and pressure measurement setup was build...... comprising this autoclave. A second high temperature and pressure measurement setup was build based on experiences from the first setup in order to perform automatized measurements. The conductivity of aqueous KOH at elevated temperatures and high concentrations was investigated using the van der Pauw method...

  14. Comparative Evaluation of Agroindustrial Byproducts for the Production of Alkaline Protease by Wild and Mutant Strains of Bacillus subtilis in Submerged and Solid State Fermentation

    OpenAIRE

    Hamid Mukhtar; Ikramul Haq

    2013-01-01

    The present study describes the screening of different agroindustrial byproducts for enhanced production of alkaline protease by a wild and EMS induced mutant strain of Bacillus subtilis IH-72EMS8. During submerged fermentation, different agro-industrial byproducts were tested which include defatted seed meals of rape, guar, sunflower, gluten, cotton, soybean, and gram. In addition to these meals, rice bran, wheat bran, and wheat flour were also evaluated for protease production. Of all the b...

  15. 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.

  16. Statistical medium optimization of an alkaline protease from Pseudomonas aeruginosa MTCC 10501, its characterization and application in leather processing.

    Science.gov (United States)

    Boopathy, Naidu Ramachandra; Indhuja, Devadas; Srinivasan, Krishnan; Uthirappan, Mani; Gupta, Rishikesh; Ramudu, Kamini Numbi; Chellan, Rose

    2013-04-01

    Proteases are shown to have greener mode of application in leather processing for dehairing of goat skins and cow hides. Production of protease by submerged fermentation with potent activity is reported using a new isolate P. aeruginosa MTCC 10501. The production parameters were optimized by statistical methods such as Plackett-Burman and response surface methodology. The optimized production medium contained (g/L); tryptone, 2.5; yeast extract, 3.0; skim milk 30.0; dextrose 1.0; inoculum concentration 4%: initial pH 6.0; incubation temperature 30 degrees C and optimum production at 48 h with protease activity of 7.6 U/mL. The protease had the following characteristics: pH optima, 9.0; temperature optima 50 degrees C; pH stability between 5.0-10.0 and temperature stability between 10-40 degrees C. The protease was observed to have high potential for dehairing of goat skins in the pre- tanning process comparable to that of the chemical process as evidenced by histology. The method offers cleaner processing using enzyme only instead of toxic chemicals in the pre-tanning process of leather manufacture.

  17. High-level expression of alkaline protease using recombinant ...

    African Journals Online (AJOL)

    AJL

    2012-02-16

    Feb 16, 2012 ... 3Department of Biotechnology and Food Technology, Faculty of Applied Sciences, Durban University of Technology, ... was composed of 0.5% yeast extract, 1% citric acid, 0.33% sodium citrate, 0.26% ..... Nucleic acids. Res.

  18. PRODUCTION AND PARTIAL CHARACTERIZATION OF ALKALINE PROTEASE FROM BACILLUS TEQUILENSIS STRAINS CSGAB0139 ISOLATED FROM SPOILT COTTAGE CHEESE

    Directory of Open Access Journals (Sweden)

    Aruna K

    2014-09-01

    Full Text Available An alkaline protease producing strain was isolated from spoilt cottage cheese sample which was identified as Bacillus tequilensis strain SCSGAB0139 on the basis of morphological, cultural, biochemical characteristics and 16S rRNA sequence analysis. Primary screening for protease production was carried out by observing for zone of hydrolysis on skim milk agar, GYEA milk agar and gelatin agar plates. Physicochemical parameters like pH of the medium, incubation time and temperature, aeration and composition of the medium were optimized for maximum protease production by this isolate. Maximum yield of protease (85.67U/ml was obtained in a medium containing peptone (5% w/v, maltose (5% w/v and KNO3, 0.5%; K2HPO4, 0.4%; trisodium citrate, 0.4; CaCl2, 0.0002%; MgSO4·7H2O, 0.05%; Na2CO3, 1%.; 1% (v/v of a trace element solution (NH46MO7O24, 0.01%; FeSO4·7H2O, 0.2%; CuSO4·5H2O, 0.02%; ZnCl2, 0.02% having pH 10, inoculated with 1%(v/v of pre-grown cell mass and incubated at 30°C on a rotary shaker (100rpm for 48hrs. Absence of any one of the following salts viz. KNO3, K2HPO4, tri-sodium citrate; MgSO4, CaCl2 and Na2CO3 from optimized medium reduced the protease production by 80% to 40%. The enzyme has an optimum pH of 9 and maintained its stability over a broad pH range between 6 and 10. Its optimum temperature is 30°C, and exhibited a stability of up to 65°C. Among metal ions only Ca2+ and Mg2+ions enhanced the enzyme activity up to 105% and 107% respectively while other metal ions reduced the activity by 40% where as EDTA exhibited the least inhibitory effect upon the enzyme. Protease activity was enhanced in the presence of isopropanol and marginally reduced in the presence of other organic solvents studied. The crude enzyme showed stability towards various surfactants such as Tween-20, Tween- 80, SDS and Triton X-100. It also showed excellent stability and compatibility with commonly used laundry detergents (Ariel, Surf excel and Surf Blue. The

  19. SHG10 keratinolytic alkaline protease from Bacillus licheniformis SHG10 DSM 28096: Robust stability and unusual non-cumbersome purification.

    Science.gov (United States)

    Embaby, Amira M; Saeed, Hesham; Hussein, Ahmed

    2016-12-01

    Present study underlines an unusual non-cumbersome-powerful strategy for purification of SHG10 keratinolytic alkaline protease from Bacillus licheniformis SHG10 DSM 28096 with robust stability properties. The enzyme was impressively purified to homogeneity with specific activity, purification fold, and yield of 613.82 U mg(-1) , 58.91 and 99%, respectively, via a sequential two-step purification strategy: precipitation with 65% (NH4 )2 SO4 and flow through fractions of DEAE-cellulose DE 53 column. SDS-PAGE conferred a monomeric enzyme with a molecular mass of 30.4 kDa. The enzyme demonstrated optimal activity at pH (10.0-11.0) and at 65 °C. It exhibited full stability at pH (6.0-11.0) over 38 h at 4 °C and at 65 °C for 15 min. Remarkable enhanced enzyme activity (130.15 and 126.37%) was retained in presence of commercial laundry detergents Oxi and Ariel after 1 h, respectively. Organic solvent stability of the enzyme was verified in butanol, ether, acetonitrile, isopropanol, and chloroform. Imposingly, full storage stability (100%) of the enzyme along 1 year in -20 °C was confirmed. Km -Vmax was 0.00174 mM-534.2 mM Sub · min(-1)  · mg protein(-1) and 1.266 mg-28.89 mg Sub · h(-1)  · mg protein(-1) on N-Suc-Ala-Ala-Pro-Phe-pNA and keratin azure, respectively. Robust stability properties of SHG10 keratinolytic alkaline protease along with rapid-efficient purification underpin its potential commercialization for industrial exploitation.

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

    Science.gov (United States)

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

    2012-01-01

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

  1. High temperature and pressure alkaline electrolysis

    DEFF Research Database (Denmark)

    Allebrod, Frank; Chatzichristodoulou, Christodoulos; Mogensen, Mogens Bjerg

    2013-01-01

    and pressures. Two measurement systems were built to perform measurements under high pressures and at elevated temperatures of up to 95 bar and 250 °C, respectively. The conductivity of aqueous KOH and aqueous KOH immobilized in a porous SrTiO3 structure were investigated at elevated temperatures and high...... the operational temperature and pressure to produce pressurized hydrogen at high rate (m3 H2·h-1·m-2 cell area) and high electrical efficiency. This work describes an exploratory technical study of the possibility to produce hydrogen and oxygen with a new type of alkaline electrolysis cell at high temperatures...... concentrations of the electrolyte using the van der Pauw method in combination with electrochemical impedance spectroscopy (EIS). Conductivity values as high as 2.9 S cm-1 for 45 wt% KOH aqueous KOH and 0.84 S cm-1 for the immobilized KOH of the same concentration were measured at 200 °C. Porous SrTiO3 was used...

  2. Microbial thiocyanate utilization under highly alkaline conditions.

    Science.gov (United States)

    Sorokin, D Y; Tourova, T P; Lysenko, A M; Kuenen, J G

    2001-02-01

    Three kinds of alkaliphilic bacteria able to utilize thiocyanate (CNS-) at pH 10 were found in highly alkaline soda lake sediments and soda soils. The first group included obligate heterotrophs that utilized thiocyanate as a nitrogen source while growing at pH 10 with acetate as carbon and energy sources. Most of the heterotrophic strains were able to oxidize sulfide and thiosulfate to tetrathionate. The second group included obligately autotrophic sulfur-oxidizing alkaliphiles which utilized thiocyanate nitrogen during growth with thiosulfate as the energy source. Genetic analysis demonstrated that both the heterotrophic and autotrophic alkaliphiles that utilized thiocyanate as a nitrogen source were related to the previously described sulfur-oxidizing alkaliphiles belonging to the gamma subdivision of the division Proteobacteria (the Halomonas group for the heterotrophs and the genus Thioalkalivibrio for autotrophs). The third group included obligately autotrophic sulfur-oxidizing alkaliphilic bacteria able to utilize thiocyanate as a sole source of energy. These bacteria could be enriched on mineral medium with thiocyanate at pH 10. Growth with thiocyanate was usually much slower than growth with thiosulfate, although the biomass yield on thiocyanate was higher. Of the four strains isolated, the three vibrio-shaped strains were genetically closely related to the previously described sulfur-oxidizing alkaliphiles belonging to the genus Thioalkalivibrio. The rod-shaped isolate differed from the other isolates by its ability to accumulate large amounts of elemental sulfur inside its cells and by its ability to oxidize carbon disulfide. Despite its low DNA homology with and substantial phenotypic differences from the vibrio-shaped strains, this isolate also belonged to the genus Thioalkalivibrio according to a phylogenetic analysis. The heterotrophic and autotrophic alkaliphiles that grew with thiocyanate as an N source possessed a relatively high level of cyanase

  3. Scale-up of an alkaline protease from Bacillus pumilus MTCC 7514 utilizing fish meal as a sole source of nutrients.

    Science.gov (United States)

    Gupta, Rishikesh Kumar; Prasad, Dinesh; Sathesh, Jaykumar; Naidu, Ramachandra Boopathy; Kamini, Numbi Ramudu; Palanivel, Saravanan; Gowthaman, Marichetti Kuppuswami

    2012-09-01

    Fish meal grades SL1 and SL2 from Sardine (Sardinella longiceps) and NJ from Pink Perch (Nemipterus japonicas) were evaluated as a sole source of carbon and nitrogen in the medium for alkaline protease production by Bacillus pumilus MTCC 7514. The analysis of the fish meal suggests that the carbon and nitrogen contents in fish meal are sufficient to justify its choice as replacement for other nutrients. Protease production increased significantly (4,914 U/ml) in medium containing only fish meal, compared with the basal medium (2,646 U/ml). However, the elimination of inorganic salts from media reduced the protease productivity. In addition, all the three grades of fish meal yielded almost the same amounts of protease when employed as the sole source of carbon and nitrogen. Nevertheless, the best results were observed in fish meal SL1 medium. Furthermore, protease production was enhanced to 6,966 U/ml and 7,047 U/ml on scaling up from flask (4,914 U/ml) to 3.7 and 20 L fermenters, respectively, using fish meal (10 g/l). Similarly, the corresponding improvement in productivities over flask (102.38 U/ml/h) was 193.5 and 195.75 U/ml/h in 3.7 and 20 L fermenters, respectively. The crude protease was found to have dehairing ability in leather processing, which is bound to have great environmental benefits.

  4. ALKALINE PROTEASE, AMYLASE AND CELLULASE ACTIVITIES OF YELLOW RASBORA, Rasbora lateristriataBlkr., AT DIFFERENT FEEDING LEVELS

    Directory of Open Access Journals (Sweden)

    Untung Susilo

    2016-11-01

    Full Text Available Alkaline proteases, amylase and cellulase activities of digestive organ of yellow rasbora, Rasbora lateristriata Blkr., was evaluated with four different feeding levels of 0.34g protein+0.03g fiber, 1.01g protein+0.10g fiber, 1.69g protein+0.16g fiber and 2.36g protein+0.23g fiber/day/100 g fish biomass. A total of 280 fish with average body weight of 0.71±0.06g were used in this study. The results showed that the difference in the feeding levels resulted in a significant difference in trypsin and chymotrypsin  activities (P .05. In conclusion, protein digestion capacity increased, but not to the digestion of starch and fiber in response to different feeding levels and the optimal feeding level for yellow rasbora was 1,01g protein+0,10g fiber/day/100 g fish biomass.

  5. Identification of a Bacillus strain producing alkaline protease%产碱性蛋白酶芽孢杆菌的鉴定

    Institute of Scientific and Technical Information of China (English)

    黄继翔

    2011-01-01

    通过测量比较在碱性蛋白平板上产生的蛋白水解圈直径,从土壤中筛选到一株高产蛋白酶菌株Bacillus sp.HFBL0079,根据生理生化特性,16S rDNA序列,鉴定为B.amyloliquefaciens.其最适培养温度为35℃-37℃,最适生长pH 8.0,在特定培养条件下16 h达到稳定期,菌体生长和蛋白酶合成同步进行.以大豆分离蛋白为氮源时发酵液具有最高酶活.发酵液在pH 10时具有最高酶活,表明为碱性蛋白酶.该菌株产生的碱性蛋白酶可水解多种天然蛋白质,对胶原蛋白水解度高于其他蛋白质,对羽毛角蛋白也有一定水解能力,提示该酶具有一定新颖性.%A protease producing spore-forming strain named HFBL0079 was islated from soil by measuring clearing zone in alkaline casein plate. Bacillus sp. HFBL0079 was identified as B. amyloliquefaciens by phenotypic properties and 16S rDNA sequence. Its optimal growth temperature was 35 ℃-37 ℃ and initial pH was 8.0. Protease activity increased quickly in log phase in accord with biomass and was steady in stationary phase, the maximal value was found in 16 hours. The highest protease activity was found when nitrogen source was soy protein isolate. The protease was alkaline protease because the highest activity was shown in pH 10. The alkaline protease was shown activity to multi substrate, the highest hydrolysis degree was found in collagen (42.3%) more than casein, oval bumin, BSA, and the hydrolysis degree of keratin was 15.3%, these results indicated the alkaline protease had some novel properties.

  6. Alkaline protease and its application in soybean peptide preparation%碱性蛋白酶及其在大豆肽制备中的应用

    Institute of Scientific and Technical Information of China (English)

    孙倩; 陈复生; 丁长河; 刘伯业

    2011-01-01

    Alkaline protease,which is an important kind of industrial enzyme,has been widely applied in food industry,medical treatment,detergent industry,leather producing and other fields.Nowadays,the enzymes for food industry mainly come from microorganism,and the effect of alkaline protease is much better.This article summarizes the producing strains,structure and properties,current use and research status of alkaline protease.Its application in soybean peptide preparation has also been mentioned.%碱性蛋白酶是一类重要的工业用酶,广泛应用于食品、医药、洗涤剂和皮革等领域。目前食品工业用酶主要来源于微生物,且实际生产中碱性蛋白酶的效果较好。从碱性蛋白酶的产生菌株、结构和性质、应用研究现状及其在大豆肽制备中的应用等方面进行了概述。

  7. Catalysis and stability of an alkaline protease from a haloalkaliphilic bacterium under non-aqueous conditions as a function of pH, salt and temperature.

    Science.gov (United States)

    Pandey, Sandeep; Rakholiya, Kalpna D; Raval, Vikram H; Singh, Satya P

    2012-09-01

    A haloalkaliphilic bacterium, isolated from Coastal Gujarat (India) was identified as Oceanobacillus sp. (GQ162111) based on 16S rRNA gene sequence. The organism grew and secreted extra cellular protease in presence of various organic solvents. At 30% (v/v) concentration of hexane, heptane, isooctane, dodecane and decane, significant growth and protease production was evident. The alkaline protease was purified in a single step on phenyl sepharose 6 FF with 28% yield. The molecular mass as judged by SDS-PAGE was 30 kDa. The temperature optimum of protease was 50°C and the enzyme retained 70% activity in 10% (v/v) isooctane. Effect of salt and pH was investigated in combination to assess the effect of isooctane. In organic solvents, the enzyme was considerably active at pH 8-11, with optimum activity at pH 10. Salt at 2 M was optimum for activity and enzyme maintained significant stability up to 18 h even at 3 M salt concentration. Patters of growth, protease production, catalysis and stability of the enzyme are presented. The study resumes significance as limited information is available on the interaction of haloalkaliphilic bacteria and their enzymes with organic solvents.

  8. Purification and characterization of a thermo- and organic solvent-tolerant alkaline protease from Bacillus sp. JER02.

    Science.gov (United States)

    Badoei-Dalfard, Arastoo; Karami, Zahra; Ravan, Hadi

    2015-01-01

    Bacillus sp. JER02 is a bacterial strain that can be grown in a medium containing organic solvents and produce a protease enzyme. JER02 protease was purified with a yield of 31.9% of total protein and 328.83-fold purification. Km and Vmax of this protease were established as 0.826 µM and 7.18 µmol/min, respectively. JER02 protease stability was stimulated about 80% by cyclohexane. It exhibited optimum temperature activity at 70°C. Furthermore, this enzyme was active in a wide range of pH (4-12) and showed maximum activity at pH 9.0. The nonionic detergents Tween-20 and Triton X-100 improved the protease activity by 30 and 20%, respectively. In addition, this enzyme was shown to be very stable in the presence of strong anionic surfactants and oxidizing agents, since it retained 77%, 93%, and 98% of its initial activity, after 1 hr of incubation at room temperature with sodium dodecyl sulfate (SDS), sodium perborate (1%, v/v) and H2O2 (1%, v/v), respectively. Overall, the unique properties of the Bacillus sp. JER02 protease suggested that this thermo- and detergent-stable, solvent-tolerant protease has great potential for industrial applications.

  9. Anticipation of Artemia sp. supply in the larviculture of the barber goby Elacatinus figaro (Gobiidae: Teleostei influenced growth, metamorphosis and alkaline protease activity

    Directory of Open Access Journals (Sweden)

    Maria Fernanda da Silva-Souza

    2015-09-01

    Full Text Available The barber goby Elacatinus figaro is considered endangered due to overexploitation by the ornamental industry. Farming marine ornamental fishes, especially the threatened ones, can be one of the measures to minimize the pressure on the natural stocks. Among the priority issues for their production is the determination of the most appropriate feeding management. The feeding protocol commonly used in the larviculture of barber goby, when the start of Artemia sp. offer occurred at the 18th DAH (days after hatching (treatment T18, was modified, by anticipating brine shrimp supply in 6 days (treatment T12. Alkaline proteases activity, growth and metamorphosis of larvae were evaluated in both protocols. Juveniles at T12 showed higher weight (0.04 ± 0.001 g and lower activity of total alkaline proteases (1.3 ± 0.2 mU mg-1 protein compared to T18 (0.02 ± 0.001 g; 2.8 ± 0.4 mU mg-1 protein, respectively. With anticipation of brine shrimp, the commencing and end of larval transformation was observed earlier (at 24 and 34 DAH, respectively in comparison to those with the supply of Artemia sp. at 18 DAH (27 and 41 DAH, respectively. Thus, the Artemia sp. anticipation was beneficial during the larviculture of the barber goby, considering that larvae reached metamorphosis earlier.

  10. Optimum conditions of enzymatic hydrolysis of rice residue protein by alkaline protease%米渣蛋白碱性蛋白酶酶解条件优化

    Institute of Scientific and Technical Information of China (English)

    许尨; 袁江兰; 靳艳; 邓川; 李文竹

    2012-01-01

    Rice dreg is the quality of protein resources. This by-product from rice residue as raw material starch sugar enterprise, degree of hydrolysis and foaming index for evaluation, of enzymatic hydrolysis of rice residue protein by alkaline protease, first examine the enzyme adds volume, pH, substrate concentration, and other factors influence on the degree of hydrolysis of rice residue protein, and then came to the best enzymatic hydrolysis by response surface test conditions. The results show that the optimal conditions enzyme solution is substrate concentration 6%, enzyme solution time 90 min, add enzyme quantity 2%, pH 8.5, enzyme solution temperature 55 %, the conditions under which get enzyme solution liquid foaming ability high.%米渣是优质的蛋白质资源。以淀粉糖企业副产品米渣为原料,以水解度和起泡性为评价指标,利用碱性蛋白酶对米渣蛋白进行酶解,首先研究酶添加量、pH、底物浓度等因素对米渣蛋白质水解度的影响,然后通过响应面试验得出最佳酶解条件。结果表明,酶解最适条件是底物浓度6%、酶解时间90min、加酶量2%、pH为8.5、酶解温度55℃,在此条件下得到的酶解液起泡性高。

  11. A novel alkaline protease with antiproliferative activity from fresh fruiting bodies of the toxic wild mushroom Amanita farinosa.

    Science.gov (United States)

    Sun, Jian; Zhao, Yongchang; Chai, Hongmei; Wang, Hexiang; Ng, Tzi Bun

    2011-01-01

    A novel protease with a molecular mass of 15 kDa was purified from fresh fruiting bodies of the wild mushroom Amanita farinosa. The purification protocol entailed anion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, cation exchange chromatography on SP-Sepharose, and gel filtration by fast protein liquid chromatography on Superdex 75. The protease was unadsorbed on DEAE-cellulose but adsorbed on Affi-gel blue gel and SP-Sepharose. It demonstrated a single 15-kDa band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS/PAGE) and a 15-kDa peak in gel filtration. The optimal pH and optimal temperature of the protease were pH 8.0 and 65 °C, respectively. Proliferation of human hepatoma HepG2 cells was inhibited by the protease with an IC(50) of 25 µM. The protease did not have antifungal or ribonuclease activity.

  12. Secretion of an alkaline protease from a salt- tolerant and alkaliphilic, Streptomyces clavuligerus strain Mit-1 Secreção de uma protease alcalina por uma cepa halotolerante e alcalifílica de Streptomyces clavuligerus, Mit-1

    Directory of Open Access Journals (Sweden)

    Jignasha T. Thumar

    2007-12-01

    Full Text Available An alkaliphilic and salt- tolerant actinomycete, Streptomyces clavuligerus strain Mit-1, was isolated from Mithapur, the western coast of India. The organism was Gram-positive, having filamentous, long thread like structure. The sporulation started after two days of growth and the optimum level of alkaline protease (130 U/ml was produced during the early stationary phase. The strain could grow and produce protease with 0-10% NaCl (w/v, the optimum being 5% NaCl (w/v. Growth and protease production was optimum at pH 9 with substantial decline at neutral pH. Sucrose and gelatin were the best carbon and nitrogen sources respectively, whereas gelatin broth was the preferred medium for protease production. Mit-1 produced substantial protease with various amino acids, when employed as the sole nitrogen sources. Crude substrates, such as molasses, whey and wheat flour had significant effect on enzyme production. The results are quite valuable, as only few actinomycetes, particularly salt-tolerant alkaliphilic ones, have so far been explored for their enzymatic potential and process optimization.Uma cepa halotolerante e alcalifílica de Streptomyces clavuligerus, Mit-1, foi isolada em Mithapur, na costa oeste da Índia. Esse microrganismo é Gram positivo e apresenta estrutura filamentosa na forma de longas cordas. A esporulação iniciou após dois dias de cultivo e o nível ótimo de produção de protease alcalina (130 U/ml foi atingido no início da fase estacionária de crescimento. A cepa foi capaz de multiplicar com 0-10% NaCl (w/v, com um ótimo de 5% NaCl (w/v. O ótimo de crescimento e produção de protease foi atingido em pH 9, apresentando declínio substancial em pH neutro. Sacarose e gelatina foram as melhores fones de carbono e nitrogênio, respectivamente, enquanto o caldo gelatina foi o melhor meio para produção de protease. A cepa Mit-1 produziu bastante protease quando vários aminoácidos foram empregados como única fonte de

  13. High-performance alkaline polymer electrolyte for fuel cell applications

    Energy Technology Data Exchange (ETDEWEB)

    Pan, Jing; Lu, Shanfu; Li, Yan; Huang, Aibin; Zhuang, Lin; Lu, Juntao [College of Chemistry and Molecular Sciences, Hubei Key Lab. of Electrochemical Power Sources, Wuhan University (China)

    2010-01-22

    Although the proton exchange membrane fuel cell (PEMFC) has made great progress in recent decades, its commercialization has been hindered by a number of factors, among which is the total dependence on Pt-based catalysts. Alkaline polymer electrolyte fuel cells (APEFCs) have been increasingly recognized as a solution to overcome the dependence on noble metal catalysts. In principle, APEFCs combine the advantages of and alkaline fuel cell (AFC) and a PEMFC: there is no need for noble metal catalysts and they are free of carbonate precipitates that would break the waterproofing in the AFC cathode. However, the performance of most alkaline polyelectrolytes can still not fulfill the requirement of fuel cell operations. In the present work, detailed information about the synthesis and physicochemical properties of the quaternary ammonia polysulfone (QAPS), a high-performance alkaline polymer electrolyte that has been successfully applied in the authors' previous work to demonstrate an APEFC completely free from noble metal catalysts (S. Lu, J. Pan, A. Huang, L. Zhuang, J. Lu, Proc. Natl. Acad. Sci. USA 2008, 105, 20611), is reported. Monitored by NMR analysis, the synthetic process of QAPS is seen to be simple and efficient. The chemical and thermal stability, as well as the mechanical strength of the synthetic QAPS membrane, are outstanding in comparison to commercial anion-exchange membranes. The ionic conductivity of QAPS at room temperature is measured to be on the order of 10{sup -2} S cm{sup -1}. Such good mechanical and conducting performances can be attributed to the superior microstructure of the polyelectrolyte, which features interconnected ionic channels in tens of nanometers diameter, as revealed by HRTEM observations. The electrochemical behavior at the Pt/QAPS interface reveals the strong alkaline nature of this polyelectrolyte, and the preliminary fuel cell test verifies the feasibility of QAPS for fuel cell applications. (Abstract Copyright [2010

  14. Production of an alkaline protease using Bacillus pumilus D3 without inactivation by SDS, its characterization and purification.

    Science.gov (United States)

    Özçelik, Burçin; Aytar, Pınar; Gedikli, Serap; Yardımcı, Ezgi; Çalışkan, Figen; Çabuk, Ahmet

    2014-06-01

    Abstract In this study, protease-producing capacity of Bacillus pumilus D3, isolated from hydrocarbon contaminated soil, was evaluated and optimized. Optimum growing conditions for B. pumilus D3 in terms of protease production were determined as 1% optimum inoculum size, 35 °C temperature, 11 pH and 48 h incubation time, respectively. Stability studies indicated that the mentioned protease was stable within the pH range of 7-10.5 and between 30 °C and 40 °C temperatures. Surprisingly, the activity of the enzyme increased in the presence of SDS with concentration up to 5 mM. The protease was concentrated 1.6-fold with ammonium sulfate precipitation and dialysis. At least six protein bands were obtained from dialysate by electrophoresis. Four clear protein bands with caseinolytic activity were detected by zymography. Dialysate was further purified by anion-exchange chromatography and the caseinolytic active fraction showed a single band between 29 and 36 kDa of reducing conditions.

  15. Production and estimation of alkaline protease by immobilized Bacillus licheniformis isolated from poultry farm soil of 24 Parganas and its reusability

    Directory of Open Access Journals (Sweden)

    Shamba Chatterjee

    2015-01-01

    Full Text Available Microbial alkaline protease has become an important industrial and commercial biotech product in the recent years and exerts major applications in food, textile, detergent, and pharmaceutical industries. By immobilization of microbes in different entrapment matrices, the enzyme produced can be more stable, pure, continuous, and can be reused which in turn modulates the enzyme production in an economical manner. There have been reports in support of calcium alginate and corn cab as excellent matrices for immobilization of Bacillus subtilis and Bacillus licheniformis, respectively. This study has been carried out using calcium alginate, κ-carrageenan, agar-agar, polyacrylamide gel, and gelatin which emphasizes not only on enzyme activity of immobilized whole cells by different entrapment matrices but also on their efficiency with respect to their reusability as first attempt. Gelatin was found to be the best matrix among all with highest enzyme activity (517 U/ml at 24 h incubation point and also showed efficiency when reused.

  16. Inducible polymerization and two-dimensional assembly of the repeats-in-toxin (RTX) domain from the Pseudomonas aeruginosa alkaline protease.

    Science.gov (United States)

    Zhang, Liang; Franks, Jonathon; Stolz, Donna B; Conway, James F; Thibodeau, Patrick H

    2014-10-21

    Self-assembling proteins represent potential scaffolds for the organization of enzymatic activities. The alkaline protease repeats-in-toxin (RTX) domain from Pseudomonas aeruginosa undergoes multiple structural transitions in the presence and absence of calcium, a native structural cofactor. In the absence of calcium, this domain is capable of spontaneous, ordered polymerization, producing amyloid-like fibrils and large two-dimensional protein sheets. This polymerization occurs under near-physiological conditions, is rapid, and can be controlled by regulating calcium in solution. Fusion of the RTX domain to a soluble protein results in the incorporation of engineered protein function into these macromolecular assemblies. Applications of this protein sequence in bacterial adherence and colonization and the generation of biomaterials are discussed.

  17. Nanoplatforms for highly sensitive fluorescence detection of cancer-related proteases.

    Science.gov (United States)

    Wang, Hongwang; Udukala, Dinusha N; Samarakoon, Thilani N; Basel, Matthew T; Kalita, Mausam; Abayaweera, Gayani; Manawadu, Harshi; Malalasekera, Aruni; Robinson, Colette; Villanueva, David; Maynez, Pamela; Bossmann, Leonie; Riedy, Elizabeth; Barriga, Jenny; Wang, Ni; Li, Ping; Higgins, Daniel A; Zhu, Gaohong; Troyer, Deryl L; Bossmann, Stefan H

    2014-02-01

    Numerous proteases are known to be necessary for cancer development and progression including matrix metalloproteinases (MMPs), tissue serine proteases, and cathepsins. The goal of this research is to develop an Fe/Fe3O4 nanoparticle-based system for clinical diagnostics, which has the potential to measure the activity of cancer-associated proteases in biospecimens. Nanoparticle-based "light switches" for measuring protease activity consist of fluorescent cyanine dyes and porphyrins that are attached to Fe/Fe3O4 nanoparticles via consensus sequences. These consensus sequences can be cleaved in the presence of the correct protease, thus releasing a fluorescent dye from the Fe/Fe3O4 nanoparticle, resulting in highly sensitive (down to 1 × 10(-16) mol l(-1) for 12 proteases), selective, and fast nanoplatforms (required time: 60 min).

  18. Processing Proteases

    DEFF Research Database (Denmark)

    Ødum, Anders Sebastian Rosenkrans

    Processing proteases are proteases which proteolytically activate proteins and peptides into their biologically active form. Processing proteases play an important role in biotechnology as tools in protein fusion technology. Fusion strategies where helper proteins or peptide tags are fused...... to the protein of interest are an elaborate method to optimize expression or purification systems. It is however critical that fusion proteins can be removed and processing proteases can facilitate this in a highly specific manner. The commonly used proteases all have substrate specificities to the N...... of few known proteases to have substrate specificity for the C-terminal side of the scissile bond. LysN exhibits specificity for lysine, and has primarily been used to complement trypsin in to proteomic studies. A working hypothesis during this study was the potential of LysN as a processing protease...

  19. Manganese Dioxide with High Specific Surface Area for Alkaline Battery

    Institute of Scientific and Technical Information of China (English)

    HUANG You-ju; LIN Yu-li; LI Wei-shan

    2012-01-01

    The authors reported a facile method for the synthesis of manganese dioxide without any template and catalyst at a low-temperature.The prepared sample was characterized with X-ray diffraction(XRD),scanning electron microscopy(SEM),Brunauer-Emmett-Teller(BET) surface analysis,Fourier transform infrared(FTIR) spectrometry,cyclic voltammetry,altemative current(AC) impedance test and battery discharge test.It is found that the prepared sample belongs to α-MnO2 and has a microsphere morphology and a large BET surface area.The electrochemical characterization indicates that the prepared sample displays a larger electrochemical capacitance than the commercial electrolytic manganese dioxides(EMD) in Na2SO4 solution,and exhibits larger discharge capacity than EMD,especially at a high rate discharge condition when it is used as cathode of alkaline Zn/MnO2 battery.

  20. 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. KM and Vmax values were calculated as 0.197 mg/mL and 7.29 μmol.mL(-)(1).min(-)(1), respectively.

  1. Calcium aluminate cement hydration in a high alkalinity environment

    Directory of Open Access Journals (Sweden)

    Palomo, Á.

    2009-03-01

    Full Text Available The present paper forms part of a broader research project that aims primarily to devise new cementitious products via the alkali activation of silico-aluminous materials. This work addresses the possibility of using small percentages of calcium aluminate cement (CAC as a source of reactive aluminium. For this reason, a preliminary review was needed of the behaviour of CACs in highly alkaline media (2, 8 and 12M NaOH solutions. Two, 28- and 180-day mechanical strength was determined and the reaction products were characterized with XRD and FTIR. The water-hydrated CAC was used as the control.The results obtained showed that CAC hardening took place much more slowly in highly alkaline media than in water. Nonetheless, the 28-day compressive strength obtained, ≥80MPa. As main reaction products, to ambient temperature and from the two days of cured, cubic aluminate C3AH6, and AH3 polymorphs are formed, instead of the usual hexagonal aluminatos (CAH10 and C2AH8 that are formed in the normal hydrate with water.El presente trabajo forma parte de una amplia investigación cuyo objetivo principal es el de elaborar nuevos materiales con propiedades cementantes mediante la activación alcalina de materiales de naturaleza silito-aluminosa. En estos estudios se contempla la posibilidad de utilizar pequeños porcentajes de cemento de aluminato de calcio (CAC como fuente de aluminio reactivo. Por ello inicialmente se ha estudiado el comportamiento de los CAC en medios fuertemente alcalinos (disoluciones de NaOH 2M, 8M y 12M. Se determinaron las resistencias mecánicas a 2, 28 y 180 días y se realizó una caracterización de los productos de reacción formados por DRX, FTIR. Como sistema de referencia se consideró la hidratación del CAC con agua.Los resultados obtenidos muestran que en medios fuertemente alcalinos se retrasan los procesos de rápido endurecimiento de CAC con agua. No obstante a 28 días se obtienen valores de resistencia a compresión

  2. Cloning, Expression and Hydrolysis Characteristics of Alkaline Protease Gene from Aspergillus oryzae%米曲霉碱性蛋白酶基因的克隆表达及水解特性

    Institute of Scientific and Technical Information of China (English)

    柯野; 陈丹; 李家洲; 谢明权; 罗晓春

    2012-01-01

    Alkaline proteases from Aspergillus oryzae are of efficient hydrolysis ability and preferable debittering effect to plant protein. In order to obtain a large amount of alkaline proteases for industrial applications, the alkaline protease gene from A. oryzae was cloned by using RT-PCR technique, and was transformed into Pichia pastoris KM71 strain, thus being successfully expressed. Then, the protease was used in the hydrolysis experiments of bovine insulin B-chain, soy protein and peanut protein. The results show that ( 1 ) when the protease is induced to express in a 10-L fermentor, the protease activity of the fermentation broth is up to 4100U/mL; (2) the optimum pH value and temperature of the protease are respectively 8.5 -9.5 and 50X. , and the protease is stable at a pH value of 6.0 to 10.0 below 40℃ ; (3) the protease is of extensive peptide-bond selectivity; and (4) the protease displays higher hydrolysis activity to soy and peanut protein than some commercial proteases. The above-mentioned results indicate that the recombinant protease is of potential application prospects.%米曲霉碱性蛋白酶对植物蛋白具有高效的水解能力和较好的脱苦作用.为了便于大量获得该酶以利于工业上的应用,文中通过RT-PCR克隆获得米曲霉碱性蛋白酶基因,将之转化到毕赤酵母KM71菌株中并成功表达,之后用该酶进行牛胰岛素氧化链B链及大豆和花生蛋白的水解试验.结果显示:在10L发酵罐中诱导表达时,发酵液中重组碱性蛋白酶的酶活达4100U/mL;该蛋白酶最适反应pH值和温度分别为8.5 ~9.5和50℃,在pH值为6.0 ~10.0和温度低于40℃时具有较好的稳定性;该酶具有广泛的肽键选择性;该酶对大豆和花生蛋白显示出了强于部分商品化蛋白酶的水解能力.以上结果表明:该重组蛋白酶具有较大的开发利用潜力.

  3. Activation of fly ashes by the high temperature and high alkalinity in ASR tests

    Institute of Scientific and Technical Information of China (English)

    2011-01-01

    High temperature and high alkalinity are typical testing conditions to accelerate the appraisal process of the suppressing effect of fly ashes on alkali silica reaction(ASR),but the reaction mechanism of fly ashes would be quite different under such conditions compared to the normal condition of temperature and alkalinity.To make a reasonable analysis of the suppressing effect of fly ashes,13 types of fly ashes were tested in this paper by both the accelerated mortar bar test method and the 60°C accelerated concrete prism test method.The results showed that the effect of fly ashes would be magnified under the condition of high temperature and high alkalinity.The XRD analysis showed that all the phases of fly ash could react with the hot alkaline solution except for mullite and a small amount of quartz.Fly ash could be significantly activated by the 80°C 1 mol/L NaOH solution,and form mainly C-S-H phase and P type zeolite,but its effect on inhibiting ASR was exaggerated then.According to the mortar strength test and the ASR suppressing test results,C-S-H phase contributed to mortar strength,but its amount did not decide the ASR suppressing effect of fly ash.

  4. Alkaline Earth Core Level Photoemission Spectroscopy of High-Temperature Superconductors

    Science.gov (United States)

    Vasquez, R.

    1993-01-01

    This paper examines photoemission measurements of the alkaline Earth core levels of high-temperature superconductors and related materials, models that seek to explain the large negative shifts observed relative to the corresponding alkaline Earth metals, and the effect of lattice site disorder on the core level spectra and the presence or absence of intrinsic surface peaks.

  5. Alkaline Earth Core Level Photoemission Spectroscopy of High-Temperature Superconductors

    Science.gov (United States)

    Vasquez, R.

    1993-01-01

    This paper examines photoemission measurements of the alkaline Earth core levels of high-temperature superconductors and related materials, models that seek to explain the large negative shifts observed relative to the corresponding alkaline Earth metals, and the effect of lattice site disorder on the core level spectra and the presence or absence of intrinsic surface peaks.

  6. A constructed alkaline consortium and its dynamics in treating alkaline black liquor with very high pollution load.

    Directory of Open Access Journals (Sweden)

    Chunyu Yang

    Full Text Available BACKGROUND: Paper pulp wastewater resulting from alkaline extraction of wheat straw, known as black liquor, is very difficult to be treated and causes serious environmental problems due to its high pH value and chemical oxygen demand (COD pollution load. Lignin, semicellulose and cellulose are the main contributors to the high COD values in black liquor. Very few microorganisms can survive in such harsh environments of the alkaline wheat straw black liquor. A naturally developed microbial community was found accidentally in a black liquor storing pool in a paper pulp mill of China. The community was effective in pH decreasing, color and COD removing from the high alkaline and high COD black liquor. FINDINGS: Thirty-eight strains of bacteria were isolated from the black liquor storing pool, and were grouped as eleven operational taxonomy units (OTUs using random amplified polymorphic DNA-PCR profiles (RAPD. Eleven representative strains of each OTU, which were identified as genera of Halomonas and Bacillus, were used to construct a consortium to treat black liquor with a high pH value of 11.0 and very high COD pollution load of 142,600 mg l(-1. After treatment by the constructed consortium, about 35.4% of color and 39,000 mg l(-1 (27.3% COD(cr were removed and the pH decreased to 7.8. 16S rRNA gene polymerase chain reaction denaturant gradient gel electrophoresis (PCR-DGGE and gas chromatography/mass spectrometry (GC/MS analysis suggested a two-stage treatment mechanism to elucidate the interspecies collaboration: Halomonas isolates were important in the first stage to produce organic acids that contributed to the pH decline, while Bacillus isolates were involved in the degradation of lignin derivatives in the second stage under lower pH conditions. CONCLUSIONS/SIGNIFICANCE: Tolerance to the high alkaline environment and good controllability of the simple consortium suggested that the constructed consortium has good potential for black liquor

  7. Isolation and rheological properties of tamarind seed polysaccharide from tamarind kernel powder using protease enzyme and high-intensity ultrasound.

    Science.gov (United States)

    Poommarinvarakul, Sukhum; Tattiyakul, Jirarat; Muangnapoh, Chirakarn

    2010-06-01

    The effectiveness of using protease and combinations of protease and high-intensity ultrasound for high-purity, high-yield tamarind seed polysaccharide (TSP) production was investigated. Tamarind kernel powder (TKP) suspension was treated with protease alone at 0.16, 0.48, and 0.80 units/mL and with protease-ultrasound combinations over 3 different orders of sequence (before, simultaneous with, and after protease digestion) using combinations of 0.48 units/mL protease and high-intensity ultrasound at 25% and 50% amplitude for 15 and 30 min. The long protease digestion time could produce high-purity isolated TSP, but the polysaccharide yields were lower. The polysaccharide purity and yield were highly improved, even at a shorter protease digestion time, when the protease treatment was combined with high-intensity ultrasound. The increased amplitude level and sonication time decreased the average molecular weight of the polysaccharide. The rheological properties of the TKP and the isolated TSP, from nondestructive oscillatory measurements, demonstrated that the latter present a viscoelastic solution. The decreasing of protein content resulted in better elasticity of the solution. The power law model could be used to fit the down curve between shear rate and shear stress data. The consistency coefficient (K) increased while the flow behavior index decreased with the increased purity of the polysaccharide as a result of increasing increased digestion time, enzyme concentration, sonication power, and sonication time.

  8. Characterization and stability of extracellular alkaline proteases from halophilic and alkaliphilic bacteria isolated from saline habitat of coastal Gujarat, India Caracterização e estabilidade de proteases alcalinas extracelulares de bactérias halofílicas e alcalifílicas isoladas de habitat salino de Gujarat, Índia

    Directory of Open Access Journals (Sweden)

    Mital S. Dodia

    2006-09-01

    Full Text Available The present study deals with the isolation and characterization of the moderately halophilic-alkaliphilic bacteria from a saline habitat in western India. Eight different bacterial strains were isolated using enrichment techniques at 20% (w/v NaCl and pH 10. The isolates exhibited diversity towards gram's reaction, colony and cell morphology. They were able to grow and produce alkaline protease over a broad range of NaCl, 5-20% (w/v and pH, 8-10. None of the isolates could grow at pH 7, and one could not grow even at pH 8. Crude and partially purified proteases from strain S5 were subjected to characterization with reference to pH, salt stability and protein folding. Optimum protease activity and stability was recorded at 10% salt and pH 9-9.5. Denaturation kinetics of S5 alkaline protease along with a reference protease was studied at 8M urea followed by renaturation. The S5 alkaline protease could be partially renatured up to 32% of the original activity. Despite of the fact that all the 8 isolates were from the same site, they displayed significant diversity with respect to their salt requirement for growth and enzyme secretion. While the effect of pH was less demarcated on growth, the protease production was significantly affected. Isolate S5 produced substantial amount of halotolerant and alkaline protease. The activity and stability of the alkaline protease in a broader range of pH and salt would definitely make this enzyme an important candidate for various industrial applications.O presente estudo relata o isolamento e caracterização de bactérias moderadamente halofilicas e alcalífilicas de um habitat salino no oeste da Índia. Oito cepas diferentes de bactérias foram isoladas empregando técnicas de enriquecimento em NaCl a 20% (p/v e pH 10. As cepas apresentaram diversidade em relação à coloração de Gram e à morfologia das colônias e células. As cepas foram capazes de multiplicar e produzir protease alcalina em uma ampla

  9. ABT-378, a Highly Potent Inhibitor of the Human Immunodeficiency Virus Protease

    Science.gov (United States)

    Sham, Hing L.; Kempf, Dale J.; Molla, Akhteruzammen; Marsh, Kennan C.; Kumar, Gondi N.; Chen, Chih-Ming; Kati, Warren; Stewart, Kent; Lal, Ritu; Hsu, Ann; Betebenner, David; Korneyeva, Marina; Vasavanonda, Sudthida; McDonald, Edith; Saldivar, Ayda; Wideburg, Norm; Chen, Xiaoqi; Niu, Ping; Park, Chang; Jayanti, Venkata; Grabowski, Brian; Granneman, G. Richard; Sun, Eugene; Japour, Anthony J.; Leonard, John M.; Plattner, Jacob J.; Norbeck, Daniel W.

    1998-01-01

    The valine at position 82 (Val 82) in the active site of the human immunodeficiency virus (HIV) protease mutates in response to therapy with the protease inhibitor ritonavir. By using the X-ray crystal structure of the complex of HIV protease and ritonavir, the potent protease inhibitor ABT-378, which has a diminished interaction with Val 82, was designed. ABT-378 potently inhibited wild-type and mutant HIV protease (Ki = 1.3 to 3.6 pM), blocked the replication of laboratory and clinical strains of HIV type 1 (50% effective concentration [EC50], 0.006 to 0.017 μM), and maintained high potency against mutant HIV selected by ritonavir in vivo (EC50, ≤0.06 μM). The metabolism of ABT-378 was strongly inhibited by ritonavir in vitro. Consequently, following concomitant oral administration of ABT-378 and ritonavir, the concentrations of ABT-378 in rat, dog, and monkey plasma exceeded the in vitro antiviral EC50 in the presence of human serum by >50-fold after 8 h. In healthy human volunteers, coadministration of a single 400-mg dose of ABT-378 with 50 mg of ritonavir enhanced the area under the concentration curve of ABT-378 in plasma by 77-fold over that observed after dosing with ABT-378 alone, and mean concentrations of ABT-378 exceeded the EC50 for >24 h. These results demonstrate the potential utility of ABT-378 as a therapeutic intervention against AIDS. PMID:9835517

  10. High-yield Bacillus subtilis protease production by solid-state fermentation.

    Science.gov (United States)

    Soares, Valeria F; Castilho, Leda R; Bon, Elba P S; Freire, Denise M G

    2005-01-01

    A Bacillus subtilis isolate was shown to be able to produce extracellular protease in solid-state fermentations (SSF) using soy cake as culture medium. A significant effect of inoculum concentration and physiological age on pro tease production was observed. Maximum activities were obtained for inocula consisting of exponentially growing cells at inoculum concentrations in the range of 0.7-2.0 mg g(-1). A comparative study on the influence of cultivation temperature and initial medium pH on protease production in SSF and in submerged fermentation (SF) revealed that in SSF a broader pH range (5-10), but the same optimum temperature (37 degrees C), is obtained when compared to SF. A kinetic study showed that enzyme production is associated with bacterial growth and that enzyme inactivation begins before biomass reaches a maximum level for both SF and SSF. Maximum protease activity and productivity were 960 U g(-1) and 15.4 U g-1 h-1 for SSF, and 12 U mL-1 and 1.3 U mL-1 h-1 for SF. When SSF protease activity was expressed by volume of enzyme extract, the enzyme level was 10-fold higher and the enzyme productivity 45% higher than in SF. These results indicate that this bacterial strain shows a high biotechnological potential for protease production in solid-state fermentation.

  11. Hydrogen embrittlement on {alpha}-iron in high alkaline environment

    Energy Technology Data Exchange (ETDEWEB)

    Hu, R.; Habashi, M.; Galland, J. [Ecole Central Paris, Chatenay-Malabry (France)

    1994-12-31

    The partial pressure of hydrogen in concrete`s pore is very low. This hydrogen is due to the chemical reaction between the silica fumes and the alkaline solutions filling the concrete`s pore. Silica fumes are added in the concrete to increase its compression resistance. If the hydrogen pressure is low, the risk of hydrogen embrittlement is also low. However, for constructional works destined to endure more than 50 years, is this risk negligible? To answer this question, the authors have studied the hydrogen embrittlement on {alpha}-iron in alkaline solutions, in the pH range 9.5 to 13.3, presenting the liquids found in the concrete`s pores after different aging, periods. Cathodic charging has been performed for low current densities in the range 0.25 to 90 A/m{sup 2} simulating several partial pressures of hydrogen on the {alpha}-iron surface with and without EDTA inhibitor. The deformation rate was 2.5{times}10{sup {minus}5} s{sup {minus}1}. Finally {alpha}-iron samples and tensile specimens have been immersed in a mixture of silica fumes and an alkaline solution at pH 13.3 in an autoclave during 1,000 hours with the aim to measure the outgassed quantity of hydrogen under vacuum at 600C and to measure also the hydrogen embrittlement. The main conclusions of this study are as following: (1) Hydrogen embrittlement is promoted by oxide Fe{sub 3}O{sub 4} film rupture and/or hydroxide Fe(OH){sub 2}. This mechanism is efficient for current densities equivalent to a cathodic potential lower or equal to {minus}1V/NHE. (2) Silica fumes in contact with a solution of pH 13.3 provoke hydrogen release and its diffusion into the {alpha}-iron, but this quantity is not enough to embrittle it.

  12. Evaluation of High Solids Alkaline Pretreatment of Rice Straw

    OpenAIRE

    Cheng, Yu-Shen; Zheng, Yi; Yu, Chao Wei; Dooley, Todd M.; Jenkins, Bryan M.; VanderGheynst, Jean S.

    2010-01-01

    Fresh-harvested, air-dried rice straw was pretreated at a water content of 5 g H2O/g straw using sodium hydroxide (NaOH) and compared to pretreatment at 10 g H2O/g straw by hydrated lime (Ca(OH)2). Full factorial experiments including parallel wash-only treatments were completed with both sources of alkali. The experiments were designed to measure the effects of alkaline loading and pretreatment time on delignification and sugar yield upon enzymatic hydrolysis. Reaction temperature was held c...

  13. Water-free Alkaline Polymer-inorganic Acid Complexes with High Conductivity at Ambient Temperature

    Institute of Scientific and Technical Information of China (English)

    O.V.Chervakov; M.V.Andriianova; V.V.Riabenko; A.V.Markevich; E.M.Shembel; D.Meshri

    2007-01-01

    1 Results Recently increased interest is shown to proton conducting materials based on the alkaline polymer-inorganic acid complexes that is caused by a possibility of their application as the high-temperature electrolyte systems for various electrochemical devices (fuel cells,sensors,lithium power sources etc.).Complexes of inorganic acids with the alkaline polymers (polybenzimidazoles[1],polyvinylpyridines[2]) are characterized by high ionic conductivity at ambient temperatures (up to 10-2 Ω-1·cm-1) a...

  14. Highly specific protease-based approach for detection of Porphyromonas gingivalis in diagnosis of periodontitis

    NARCIS (Netherlands)

    Kaman, W.E.; Galassi, F.; de Soet, J.J.; Bizzarro, S.; Loos, B.G.; Veerman, E.C.I.; van Belkum, A.; Hays, J.P.; Bikker, F.J.

    2012-01-01

    Porphyromonas gingivalis is associated with the development of periodontitis. Here we describe the development of a highly specific protease-based diagnostic method for the detection of P. gingivalis in gingival crevicular fluid. Screening of a proteolytic peptide substrate library, including fluoro

  15. Highly specific protease-based approach for detection of porphyromonas gingivalis in diagnosis of periodontitis

    NARCIS (Netherlands)

    J.P. Hays (John); W.E. Kaman (Wendy); F. Galassi (Fabiano); J.J. de Soet (Johannes); S. Bizzarro (Sergio); B.G. Loos (Bruno G.); E.C.I. Veerman (Enno); A.F. van Belkum (Alex); F.J. Bikkerk

    2012-01-01

    textabstractPorphyromonas gingivalis is associated with the development of periodontitis. Here we describe the development of a highly specific protease-based diagnostic method for the detection of P. gingivalis in gingival crevicular fluid. Screening of a proteolytic peptide substrate library, incl

  16. Evaluation of high solids alkaline pretreatment of rice straw.

    Science.gov (United States)

    Cheng, Yu-Shen; Zheng, Yi; Yu, Chao Wei; Dooley, Todd M; Jenkins, Bryan M; VanderGheynst, Jean S

    2010-11-01

    Fresh-harvested, air-dried rice straw was pretreated at a water content of 5 g H(2)O/g straw using sodium hydroxide (NaOH) and compared to pretreatment at 10 g H(2)O/g straw by hydrated lime (Ca(OH)(2)). Full factorial experiments including parallel wash-only treatments were completed with both sources of alkali. The experiments were designed to measure the effects of alkaline loading and pretreatment time on delignification and sugar yield upon enzymatic hydrolysis. Reaction temperature was held constant at 95 degrees C for lime pretreatment and 55 degrees C for NaOH pretreatment. The range of delignification was 13.1% to 27.0% for lime pretreatments and was 8.6% to 23.1% for NaOH pretreatments. Both alkaline loading and reaction time had significant positive effects (p pretreatment at 95 degrees C, but there was little effect observed at 55 degrees C. Post-pretreatment washing of biomass was not necessary for subsequent enzymatic hydrolysis. Maximum glucose yields were 176.3 mg/g dried biomass (48.5% conversion efficiency of total glucose) in lime-pretreated and unwashed biomass and were 142.3 mg/g dried biomass (39.2% conversion efficiency of total glucose) in NaOH-pretreated and unwashed biomass.

  17. A highly sensitive and simply operated protease sensor toward point-of-care testing.

    Science.gov (United States)

    Park, Seonhwa; Shin, Yu Mi; Seo, Jeongwook; Song, Ji-Joon; Yang, Haesik

    2016-04-21

    Protease sensors for point-of-care testing (POCT) require simple operation, a detection period of less than 20 minutes, and a detection limit of less than 1 ng mL(-1). However, it is difficult to meet these requirements with protease sensors that are based on proteolytic cleavage. This paper reports a highly reproducible protease sensor that allows the sensitive and simple electrochemical detection of the botulinum neurotoxin type E light chain (BoNT/E-LC), which is obtained using (i) low nonspecific adsorption, (ii) high signal-to-background ratio, and (iii) one-step solution treatment. The BoNT/E-LC detection is based on two-step proteolytic cleavage using BoNT/E-LC (endopeptidase) and l-leucine-aminopeptidase (LAP, exopeptidase). Indium-tin oxide (ITO) electrodes are modified partially with reduced graphene oxide (rGO) to increase their electrocatalytic activities. Avidin is then adsorbed on the electrodes to minimize the nonspecific adsorption of proteases. Low nonspecific adsorption allows a highly reproducible sensor response. Electrochemical-chemical (EC) redox cycling involving p-aminophenol (AP) and dithiothreitol (DTT) is performed to obtain a high signal-to-background ratio. After adding a C-terminally AP-labeled oligopeptide, DTT, and LAP simultaneously to a sample solution, no further treatment of the solution is necessary during detection. The detection limits of BoNT/E-LC in phosphate-buffered saline are 0.1 ng mL(-1) for an incubation period of 15 min and 5 fg mL(-1) for an incubation period of 4 h. The detection limit in commercial bottled water is 1 ng mL(-1) for an incubation period of 15 min. The developed sensor is selective to BoNT/E-LC among the four types of BoNTs tested. These results indicate that the protease sensor meets the requirements for POCT.

  18. Hydroxide Self-Feeding High-Temperature Alkaline Direct Formate Fuel Cells.

    Science.gov (United States)

    Li, Yinshi; Sun, Xianda; Feng, Ying

    2017-03-11

    Conventionally, both the thermal degradation of the anion-exchange membrane and the requirement of additional hydroxide for fuel oxidation reaction hinder the development of the high-temperature alkaline direct liquid fuel cells. The present work addresses these two issues by reporting a polybenzimidazole-membrane-based direct formate fuel cell (DFFC). Theoretically, the cell voltage of the high-temperature alkaline DFFC can be as high as 1.45 V at 90 °C. It has been demonstrated that a proof-of-concept alkaline DFFC without adding additional hydroxide yields a peak power density of 20.9 mW cm(-2) , an order of magnitude higher than both alkaline direct ethanol fuel cells and alkaline direct methanol fuel cells, mainly because the hydrolysis of formate provides enough OH(-) ions for formate oxidation reaction. It was also found that this hydroxide self-feeding high-temperature alkaline DFFC shows a stable 100 min constant-current discharge at 90 °C, proving the conceptual feasibility.

  19. Characterization of the proteases in the midgut of the xylophagous larvae of Oemona hirta (Coleoptera:Cerambycidae)

    Institute of Scientific and Technical Information of China (English)

    Brian David Shaw; John Tane Christeller

    2009-01-01

    The protein digestive capability oftbe larvae of the longhorn beetle (Oemona hirta,Coleoptera:Cerambycidae,Fabricius,1775) was investigated.This species feeds only on wood where there is a high proportion of vascular tissue.The pH of the midgut,the major digestive organ,was alkaline and protein hydrolysis was maximal at alkaline pH.Use of specific synthetic peptide substrates showed that the major protease activities were the endopeptidases,trypsin and chymotrypsin-like activity,and the exopeptidase,leucine aminopeptidase and the pH curves corresponded to that with protein substrate.Studies using a range ofsefine protease inhibitors as well as specific inhibitors ofmetalloproteases,cysteine proteases and aspartate proteases confirmed a serine protease-based digestive system similar to earlier reports of sapwood-feeding Cerambycids.Control of these insect pests using protease inhibitors is discussed.

  20. Revealing the beneficial effect of protease supplementation to high gravity beer fermentations using "-omics" techniques

    Directory of Open Access Journals (Sweden)

    Workman Chris

    2011-04-01

    Full Text Available Abstract Background Addition of sugar syrups to the basic wort is a popular technique to achieve higher gravity in beer fermentations, but it results in dilution of the free amino nitrogen (FAN content in the medium. The multicomponent protease enzyme Flavourzyme has beneficial effect on the brewer's yeast fermentation performance during high gravity fermentations as it increases the initial FAN value and results in higher FAN uptake, higher specific growth rate, higher ethanol yield and improved flavour profile. Results In the present study, transcriptome and metabolome analysis were used to elucidate the effect on the addition of the multicomponent protease enzyme Flavourzyme and its influence on the metabolism of the brewer's yeast strain Weihenstephan 34/70. The study underlines the importance of sufficient nitrogen availability during the course of beer fermentation. The applied metabolome and transcriptome analysis allowed mapping the effect of the wort sugar composition on the nitrogen uptake. Conclusion Both the transcriptome and the metabolome analysis revealed that there is a significantly higher impact of protease addition for maltose syrup supplemented fermentations, while addition of glucose syrup to increase the gravity in the wort resulted in increased glucose repression that lead to inhibition of amino acid uptake and hereby inhibited the effect of the protease addition.

  1. High-resolution structure of a retroviral protease folded as a monomer

    Energy Technology Data Exchange (ETDEWEB)

    Gilski, Miroslaw [A. Mickiewicz University, 60-780 Poznan (Poland); Polish Academy of Sciences, 61-704 Poznan (Poland); Kazmierczyk, Maciej; Krzywda, Szymon [A. Mickiewicz University, 60-780 Poznan (Poland); Zábranská, Helena [Academy of Sciences of the Czech Republic, 166 10 Prague (Czech Republic); Cooper, Seth; Popović, Zoran [University of Washington, Box 352350, Seattle, WA 98195 (United States); Khatib, Firas; DiMaio, Frank; Thompson, James; Baker, David [University of Washington, Box 357350, Seattle, WA 98195 (United States); Pichová, Iva [Academy of Sciences of the Czech Republic, 166 10 Prague (Czech Republic); Jaskolski, Mariusz, E-mail: mariuszj@amu.edu.pl [A. Mickiewicz University, 60-780 Poznan (Poland); Polish Academy of Sciences, 61-704 Poznan (Poland)

    2011-11-01

    The crystal structure of Mason–Pfizer monkey virus protease folded as a monomer has been solved by molecular replacement using a model generated by players of the online game Foldit. The structure shows at high resolution the details of a retroviral protease folded as a monomer which can guide rational design of protease dimerization inhibitors as retroviral drugs. Mason–Pfizer monkey virus (M-PMV), a D-type retrovirus assembling in the cytoplasm, causes simian acquired immunodeficiency syndrome (SAIDS) in rhesus monkeys. Its pepsin-like aspartic protease (retropepsin) is an integral part of the expressed retroviral polyproteins. As in all retroviral life cycles, release and dimerization of the protease (PR) is strictly required for polyprotein processing and virion maturation. Biophysical and NMR studies have indicated that in the absence of substrates or inhibitors M-PMV PR should fold into a stable monomer, but the crystal structure of this protein could not be solved by molecular replacement despite countless attempts. Ultimately, a solution was obtained in mr-rosetta using a model constructed by players of the online protein-folding game Foldit. The structure indeed shows a monomeric protein, with the N- and C-termini completely disordered. On the other hand, the flap loop, which normally gates access to the active site of homodimeric retropepsins, is clearly traceable in the electron density. The flap has an unusual curled shape and a different orientation from both the open and closed states known from dimeric retropepsins. The overall fold of the protein follows the retropepsin canon, but the C{sup α} deviations are large and the active-site ‘DTG’ loop (here NTG) deviates up to 2.7 Å from the standard conformation. This structure of a monomeric retropepsin determined at high resolution (1.6 Å) provides important extra information for the design of dimerization inhibitors that might be developed as drugs for the treatment of retroviral infections

  2. Production of High Amounts of Hepatotoxin Nodularin and New Protease Inhibitors Pseudospumigins by the Brazilian Benthic Nostoc sp. CENA543

    Directory of Open Access Journals (Sweden)

    Jouni Jokela

    2017-10-01

    Full Text Available Nostoc is a cyanobacterial genus, common in soils and a prolific producer of natural products. This research project aimed to explore and characterize Brazilian cyanobacteria for new bioactive compounds. Here we report the production of hepatotoxins and new protease inhibitors from benthic Nostoc sp. CENA543 isolated from a small, shallow, saline-alkaline lake in the Nhecolândia, Pantanal wetland area in Brazil. Nostoc sp. CENA543 produces exceptionally high amounts of nodularin-R. This is the first free-living Nostoc that produces nodularin at comparable levels as the toxic, bloom-forming, Nodularia spumigena. We also characterized pseudospumigins A–F, which are a novel family of linear tetrapeptides. Pseudospumigins are structurally related to linear tetrapeptide spumigins and aeruginosins both present in N. spumigena but differ in respect to their diagnostic amino acid, which is Ile/Leu/Val in pseudospumigins, Pro/mPro in spumigins, and Choi in aeruginosins. The pseudospumigin gene cluster is more similar to the spumigin biosynthetic gene cluster than the aeruginosin gene cluster. Pseudospumigin A inhibited trypsin (IC50 4.5 μM after 1 h in a similar manner as spumigin E from N. spumigena but was almost two orders of magnitude less potent. This study identifies another location and environment where the hepatotoxic nodularin has the potential to cause the death of eukaryotic organisms.

  3. Revealing the beneficial effect of protease supplementation to high gravity beer fermentations using "-omics" techniques

    DEFF Research Database (Denmark)

    Piddocke, Maya Petrova; Fazio, Alessandro; Vongsangnak, Wanwipa

    2011-01-01

    Background: Addition of sugar syrups to the basic wort is a popular technique to achieve higher gravity in beer fermentations, but it results in dilution of the free amino nitrogen (FAN) content in the medium. The multicomponent protease enzyme Flavourzyme has beneficial effect on the brewer......'s yeast fermentation performance during high gravity fermentations as it increases the initial FAN value and results in higher FAN uptake, higher specific growth rate, higher ethanol yield and improved flavour profile. Results: In the present study, transcriptome and metabolome analysis were used...... syrup to increase the gravity in the wort resulted in increased glucose repression that lead to inhibition of amino acid uptake and hereby inhibited the effect of the protease addition....

  4. Characterization of Laboratory Prepared Concrete Pastes Exposed to High Alkaline and High Sodium Salt Solutions

    Energy Technology Data Exchange (ETDEWEB)

    Langton, C. A. [Savannah River Site (SRS), Aiken, SC (United States). Savannah River National Lab. (SRNL)

    2016-06-30

    The objective of this study was to identify potential chemical degradation mechanisms for the Saltstone Disposal Unit (SDU) concretes, which over the performance life of the structures may be exposed to highly alkaline sodium salt solutions containing sulfate, hydroxide, and other potentially corrosive chemicals in salt solution and saltstone flush water, drain water, leachate and / or pore solution. The samples analyzed in this study were cement pastes prepared in the SIMCO Technologies, Inc. concrete laboratory. They were based on the paste fractions of the concretes used to construct the Saltstone Disposal Units (SDUs). SDU 1 and 4 concrete pastes were represented by the PV1 test specimens. The paste in the SDU 2, 3, 5, and 6 concrete was represented by the PV2 test specimens. SIMCO Technologies, Inc. selected the chemicals and proportions in the aggressive solutions to approximate proportions in the saltstone pore solution [2, 3, 5, and 6]. These test specimens were cured for 56 days in curing chamber before being immersed in aggressive solutions. After exposure, the samples were frozen to prevent additional chemical transport and reaction. Selected archived (retrieved from the freezer) samples were sent to the Savannah River National Laboratory (SRNL) for additional characterization using x-ray diffraction (XRD), scanning electron microscopy (SEM), and energy dispersive x-ray (EDX) spectroscopy. Characterization results are summarized in this report. In addition, a correlation between the oxide composition of the pastes and their chemical durability in the alkaline salt solutions is provided.

  5. Effect of pH and temperature on stability and kinetics of novel extracellular serine alkaline protease (70 kDa).

    Science.gov (United States)

    Bhunia, Biswanath; Basak, Bikram; Mandal, Tamal; Bhattacharya, Pinaki; Dey, Apurba

    2013-03-01

    A novel extracellular serine protease (70 kDa by SDS-PAGE) was purified and characterized. This enzyme retained more than 93% of its initial activity after preincubation for 30 min at 37 °C in the presence of 25% (v/v) tested organic solvents and showed feather degradation activity. The purified enzyme was deactivated at various combinations of pH and temperature to examine the interactive effect of them on enzyme activity. The deactivation process was modeled as first-order kinetics and the deactivation rate constant (k(d)) was found to be minimum at pH 9 and 37 °C. The kinetic analysis of enzyme over a range of pH values indicated two pK values at 6.21 and at 10.92. The lower pK value was likely due to the catalytic histidine in the free enzyme and higher pK value likely reflected deprotonation of the proline moiety of the substrate but ionization of the active site serine is another possibility. Inhibition kinetic showed that enzyme is serine protease because enzyme was competitively inhibited by antipain and aprotinin as these compounds are known to be competitive inhibitors of serine protease. The organic solvent, thermal and pH tolerances of enzyme suggested that it may have potential for use as a biocatalyst in industry.

  6. The effect of an exogenous protease on the fermentation and nutritive value of high-moisture corn.

    Science.gov (United States)

    Kung, L; Windle, M C; Walker, N

    2014-03-01

    The objective of this study was to determine if treating high-moisture corn at harvest with an exogenous protease could accelerate the increase in in vitro ruminal starch degradation that is normally found with advancing times of ensiling. Ground high-moisture corn (HMC; 73% dry matter) was untreated or treated with an exogenous protease to achieve a final concentration of 2,000 mg of protease/kg of fresh corn. Corn was ensiled in laboratory-scale bags (approximately 500 g) that were evacuated of air, heat-sealed, and stored at 22 to 23°C for 70 and 140 d. Samples of freshly treated corn samples were collected to represent d 0 samples. Treatment of HMC with protease did not affect the gross populations of lactic acid bacteria or yeasts throughout the ensiling period. Treatment of HMC with protease resulted in higher concentrations of lactic acid and ethanol after 70 but not 140 d of ensiling. Concentrations of crude protein, water-soluble carbohydrates, and starch were unaffected by treatment with protease within each sampling day. After 70 or 140 d of ensiling, HMC that was treated with protease had higher concentrations of soluble protein (as a % of crude protein) and NH3-N, and had lower concentrations of prolamin protein, compared with untreated corn. In vitro rumen degradability (7-h incubation) of starch was greater in protease-treated versus untreated corn at all sampling days but the difference was more pronounced after 70 and 140 d compared with d 0. Concentrations of soluble protein and NH3-N were positively correlated with in vitro starch degradation. Conversely, the concentrations of prolamin protein in HMC were negatively correlated with in vitro starch degradation. Treating HMC with an exogenous protease could be a method to obtain greater potential for ruminal starch fermentation after a relatively short period of ensiling. Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  7. Shifts in leaf N:P stoichiometry during rehabilitation in highly alkaline bauxite processing residue sand.

    Science.gov (United States)

    Goloran, Johnvie B; Chen, Chengrong; Phillips, Ian R; Elser, James J

    2015-10-07

    Large quantities of sodic and alkaline bauxite residue are produced globally as a by-product from alumina refineries. Ecological stoichiometry of key elements [nitrogen (N) and phosphorus (P)] plays a critical role in establishing vegetation cover in bauxite residue sand (BRS). Here we examined how changes in soil chemical properties over time in rehabilitated sodic and alkaline BRS affected leaf N to P stoichiometry of native species used for rehabilitation. Both Ca and soil pH influenced the shifts in leaf N:P ratios of the study species as supported by consistently significant positive relationships (P stoichiometry can effectively provide a meaningful assessment on understanding nutrient limitation and productivity of native species used for vegetating highly sodic and alkaline BRS, and is a crucial indicator for assessing ecological rehabilitation performance.

  8. High Temperature Alkaline Electrolysis Cells with Metal Foam Based Gas Diffusion Electrodes

    DEFF Research Database (Denmark)

    Chatzichristodoulou, Christodoulos; Allebrod, Frank; Mogensen, Mogens Bjerg

    2016-01-01

    Alkaline electrolysis cells operating at 250°C and 40 bar are able to convert electrical energy into hydrogen at very high efficiencies and power densities. In the present work we demonstrate the application of a PTFE hydrophobic network and Ag nanowires as oxygen evolution electrocatalyst...

  9. Critical parameters in cost-effective alkaline extraction for high protein yield from leaves

    NARCIS (Netherlands)

    Zhang, C.; Sanders, J.P.M.; Bruins, M.E.

    2014-01-01

    Leaves are potential resources for feed or food, but their applications are limited due to a high proportion of insoluble protein and inefficient processing. To overcome these problems, parameters of alkaline extraction were evaluated using green tea residue (GTR). Protein extraction could be maximi

  10. Surprisingly high stability of barley lipid transfer protein, LTP1, towards denaturant, heat and proteases

    DEFF Research Database (Denmark)

    Lindorff-Larsen, Kresten; Winther, J R

    2001-01-01

    Barley LTP1 belongs to a large family of plant proteins termed non-specific lipid transfer proteins. The in vivo function of these proteins is unknown, but it has been suggested that they are involved in responses towards stresses such as pathogens, drought, heat, cold and salt. Also, the proteins...... have been suggested as transporters of monomers for cutin synthesis. We have analysed the stability of LTP1 towards denaturant, heat and proteases and found it to be a highly stable protein, which apparently does not denature at temperatures up to 100 degrees C. This high stability may be important...

  11. Bacillus licheniformis proteases as high value added products from fermentation of wastewater sludge: pre-treatment of sludge to increase the performance of the process.

    Science.gov (United States)

    Drouin, M; Lai, C K; Tyagi, R D; Surampalli, R Y

    2008-01-01

    Wastewater sludge is a complex raw material that can support growth and protease production by Bacillus licheniformis. In this study, sludge was treated by different thermo-alkaline pre-treatment methods and subjected to Bacillus licheniformis fermentation in bench scale fermentors under controlled conditions. Thermo-alkaline treatment was found to be an effective pre-treatment process in order to enhance the proteolytic activity. Among the different pre-treated sludges tested, a mixture of raw and hydrolysed sludge caused an increase of 15% in the protease activity, as compared to the untreated sludge. The benefit of hydrolysis has been attributed to a better oxygen transfer due to decrease in media viscosity and to an increase in nutrient availability. Foam formation was a major concern during fermentation with hydrolysed sludge. The studies showed that addition of a chemical anti-foaming agent (polypropylene glycol) during fermentation to control foam could negatively influence the protease production by increasing the viscosity of sludge.

  12. Synthesis of High-Quality Graphene through Electrochemical Exfoliation of Graphite in Alkaline Electrolyte

    OpenAIRE

    Tripathi, Prashant; Patel, Ch. Ravi Prakash; Shaz, M. A.; Srivastava, O N

    2013-01-01

    Owing to wide variety of applications of graphene, high-quality and economical way of synthesizing graphene is highly desirable. In this study, we report a cost effective and simple approach to production of high-quality graphene. Here the synthesis route is based on electrochemical exfoliation of graphite. Instead of using strong acids (which oxidise and damage the geometrical topology of graphene), we have used alkaline solution (KOH dissolved in water) as electrolyte. TEM analysis shows th...

  13. Microbial communities associated with the anthropogenic, highly alkaline environment of a saline soda lime, Poland.

    Science.gov (United States)

    Kalwasińska, Agnieszka; Felföldi, Tamás; Szabó, Attila; Deja-Sikora, Edyta; Kosobucki, Przemysław; Walczak, Maciej

    2017-07-01

    Soda lime is a by-product of the Solvay soda process for the production of sodium carbonate from limestone and sodium chloride. Due to a high salt concentration and alkaline pH, the lime is considered as a potential habitat of haloalkaliphilic and haloalkalitolerant microbial communities. This artificial and unique environment is nutrient-poor and devoid of vegetation, due in part to semi-arid, saline and alkaline conditions. Samples taken from the surface layer of the lime and from the depth of 2 m (both having pH ~11 and ECe up to 423 dS m(-1)) were investigated using culture-based (culturing on alkaline medium) and culture-independent microbiological approaches (microscopic analyses and pyrosequencing). A surprisingly diverse bacterial community was discovered in this highly saline, alkaline and nutrient-poor environment, with the bacterial phyla Proteobacteria (representing 52.8% of the total bacterial community) and Firmicutes (16.6%) showing dominance. Compared to the surface layer, higher bacterial abundance and diversity values were detected in the deep zone, where more stable environmental conditions may occur. The surface layer was dominated by members of the genera Phenylobacterium, Chelativorans and Skermanella, while in the interior layer the genus Fictibacillus was dominant. The culturable aerobic, haloalkaliphilic bacteria strains isolated in this study belonged mostly to the genus Bacillus and were closely related to the species Bacillus pseudofirmus, B. cereus, B. plakortidis, B. thuringensis and B. pumilus.

  14. Versatile assays for high throughput screening for activators or inhibitors of intracellular proteases and their cellular regulators.

    Directory of Open Access Journals (Sweden)

    Hideki Hayashi

    Full Text Available Intracellular proteases constitute a class of promising drug discovery targets. Methods for high throughput screening against these targets are generally limited to in vitro biochemical assays that can suffer many technical limitations, as well as failing to capture the biological context of proteases within the cellular pathways that lead to their activation. METHODS #ENTITYSTARTX00026;We describe here a versatile system for reconstituting protease activation networks in yeast and assaying the activity of these pathways using a cleavable transcription factor substrate in conjunction with reporter gene read-outs. The utility of these versatile assay components and their application for screening strategies was validated for all ten human Caspases, a family of intracellular proteases involved in cell death and inflammation, including implementation of assays for high throughput screening (HTS of chemical libraries and functional screening of cDNA libraries. The versatility of the technology was also demonstrated for human autophagins, cysteine proteases involved in autophagy.Altogether, the yeast-based systems described here for monitoring activity of ectopically expressed mammalian proteases provide a fascile platform for functional genomics and chemical library screening.

  15. Oxidative Alkaline leaching of Americium from simulated high-level nuclear waste sludges

    Energy Technology Data Exchange (ETDEWEB)

    Reed, Wendy A.; Garnov, Alexander Yu.; Rao, Linfeng; Nash, Kenneth L.; Bond, Andrew H.

    2004-01-23

    Oxidative alkaline leaching has been proposed to pre-treat the high-level nuclear waste sludges to remove some of the problematic (e.g., Cr) and/or non-radioactive (e.g., Na, Al) constituents before vitrification. It is critical to understand the behavior of actinides, americium and plutonium in particular, in oxidative alkaline leaching. We have studied the leaching behavior of americium from four different sludge simulants (BiPO{sub 4}, BiPO{sub 4 modified}, Redox, PUREX) using potassium permanganate and potassium persulfate in alkaline solutions. Up to 60% of americium sorbed onto the simulants is leached from the sludges by alkaline persulfate and permanganate. The percentage of americium leached increases with [NaOH] (between 1.0 and 5.0 M). The initial rate of americium leaching by potassium persulfate increases in the order BiPO{sub 4} sludge < Redox sludge < PUREX sludge. The data are most consistent with oxidation of Am{sup 3+} in the sludge to either AmO{sub 2}{sup +} or AmO{sub 2}{sup 2+} in solution. Though neither of these species is expected to exhibit long-term stability in solution, the potential for mobilization of americium from sludge samples would have to be accommodated in the design of any oxidative leaching process for real sludge samples.

  16. 印尼热泉中产嗜热碱性蛋白酶菌株筛选及酶学性质研究%Screening and Characterization of a Crude Thermophilic Alkaline Protease From an Isolated Strain of Brevibacillus PBI in Indonesia's Hot Spring

    Institute of Scientific and Technical Information of China (English)

    王帅; 林学政; 黄晓航; 郑立; ZILDA Dewi Seswita

    2012-01-01

    采用MSM寡营养培养基从印度尼西亚Pantai cermin,Kalianda和Banyuwedang三个地区的热泉水样、泥样以及沉积物样品中分离获得细菌菌株,通过检测蛋白酶产生透明圈和福林酚蛋白酶活性测j定相结合的方法,从中筛选出1株产嗜热蛋白酶的菌株PBI,该菌株经初步鉴定为短杆菌属(Brevibacillus),并对其酶学性质进行了研究.结果表明,菌株PBI产蛋白酶的最适酶活温度为60℃,最适pH值为8.0~9.0,具有较好的热稳定性和pH稳定性.60℃时酶的半衰期为30 min,70℃条件下20 min仍保持46.1%的酶活,该酶在pH值为5.0~9.0范围的缓冲液中酶活相对稳定.其产嗜热蛋白酶的酶活力最高可达到60.53 U/mL,在100℃条件下仍能保留26.37%的相对酶活.Fe2+,Fe3+,Cu2+和Zn2+对嗜热蛋白酶活性具有明显的抑制作用.该嗜热蛋白酶对EDTA敏感,苯甲基磺酰氟(PMSF)、亮抑酶肽(Leupeptin)、苄眯(Benzamidine)和胃蛋白酶抑制剂(Pepstain A)对该嗜热蛋白酶都有一定的抑制作用,说明其酶活性受到丝氨酸、半胱氨酸残基的影响.结果表明,该菌株是1株具有进一步改造利用价值的产蛋白酶菌株.%Thertnophilic bacteria strains were isolated from water samples, soil samples and sediment samples of three Indonesia's hot spring area (Pantai cermin, Kalianda and Banyu wedang) by using MSM oligo-medium. A strain of bacterium! by detecting hydrolysis halos of protease and its activity. The protease belonged to thermophilic alkaline protease, which showed high-temperature stability and pH stability. In addition, the results revealed that the optimal temperature and pH were 60 °C and 8. 0~9. 0 for protease activity, respectively. The enzyme activity decreased faintly at 60℃ for 10 min, the enzyme stability declined with increasing temperature, the enzyme half-life is 30min at 60 °C , enzyme activity remained 46. 1 % at 70 °C for 20 min, the enzyme activity was relatively stable in the

  17. 碱性蛋白酶水解米糠蛋白动力学特性研究%Research on Kinetics Characteristics of Hydrolyzing Rice Bran Protein by Alkaline Protease

    Institute of Scientific and Technical Information of China (English)

    翟爱华; 李新华

    2012-01-01

    The response mechanism and kinetic behavior of bioactive peptides by protease were studied. Rice bran protein was used as raw material. Based on the classic Michaelis - Menten equation, the enzymatic kinetics of the system of rice bran protein and alkaline protease was researched by the method of mathematical derivation combined with experiment. The mechanism model of single substrate hydrolysis of protein and inactivation of protease were considered to build the kinetics model of R =aexp[ -b(DH) ] ,and the parameters "a" and "b" of the kinetics model were determined. Origin 8. 0 software was applied to fit the deduced formula and calculate related parameters of enzyme kinetic model. It was shown that the rate of hydrolysis kinetic model was R = (94. 754e0 - 0. 0597s0 ) exp [ -0. 157 (DH)] and the kinetic model of degree of hydrolysis and time of hydrolysis was DH = 6. 37× ln[1 +(14. 88e0/s0 -0. 009)t]. The kinetic constants of the system of rice bran protein and alkaline protease were fitted by experiments and the results showed that the constant K4 of enzyme inactivation was 16. 144 min-1 and the constant k2 of enzymatic reaction rate was 94. 754 min-1 .%研究蛋白酶水解制备生物活性多肽反应机制与动力学行为,基于经典的米氏方程理论,应用数学推导结合试验研究的方法,以米糠蛋白为原料,对米糠蛋白-碱性蛋白酶体系进行酶解动力学研究.考虑蛋白质单底物水解、蛋白酶失活的机理模型,构建动力学模型R=aexp[-b(DH)],其中对参数a值和b值进行确定.利用Origin 8.0软件,对推导出的公式进行拟合得到水解速率动力学模型为R=(94.754e0-0.0597s0)exp[-0.157(DH)],水解度-水解时间的动力学模型:DH =6.37ln[1+(14.88e0/s0-0.009)t].对于米糠蛋白-碱性蛋白酶模型体系,经试验拟合,并求得该体系动力学常数:酶失活常数K4为16.144 min-1,酶解反应速率常数k2为94.754 min-1.

  18. A facile chemical route for recovery of high quality zinc oxide nanoparticles from spent alkaline batteries.

    Science.gov (United States)

    Deep, Akash; Sharma, Amit L; Mohanta, Girish C; Kumar, Parveen; Kim, Ki-Hyun

    2016-05-01

    Recycling of spent domestic batteries has gained a great environmental significance. In the present research, we propose a new and simple technique for the recovery of high-purity zinc oxide nanoparticles from the electrode waste of spent alkaline Zn-MnO2 batteries. The electrode material was collected by the manual dismantling and mixed with 5M HCl for reaction with a phosphine oxide reagent Cyanex 923® at 250°C for 30min. The desired ZnO nanoparticles were restored from the Zn-Cyanex 923 complex through an ethanolic precipitation step. The recovered particle product with about 5nm diameter exhibited fluorescent properties (emission peak at 400nm) when excited by UV radiation (excitation energy of 300nm). Thus, the proposed technique offered a simple and efficient route for recovering high purity ZnO nanoparticles from spent alkaline batteries.

  19. Probing the crucial role of Leu31 and Thr33 of the Bacillus pumilus CBS alkaline protease in substrate recognition and enzymatic depilation of animal hide.

    Directory of Open Access Journals (Sweden)

    Nadia Zaraî Jaouadi

    Full Text Available The sapB gene, encoding Bacillus pumilus CBS protease, and seven mutated genes (sapB-L31I, sapB-T33S, sapB-N99Y, sapB-L31I/T33S, sapB-L31I/N99Y, sapB-T33S/N99Y, and sapB-L31I/T33S/N99Y were overexpressed in protease-deficient Bacillus subtilis DB430 and purified to homogeneity. SAPB-N99Y and rSAPB displayed the highest levels of keratinolytic activity, hydrolysis efficiency, and enzymatic depilation. Interestingly, and at the semi-industrial scale, rSAPB efficiently removed the hair of goat hides within a short time interval of 8 h, thus offering a promising opportunity for the attainment of a lime and sulphide-free depilation process. The efficacy of the process was supported by submitting depilated pelts and dyed crusts to scanning electron microscopic analysis, and the results showed well opened fibre bundles and no apparent damage to the collagen layer. The findings also revealed better physico-chemical properties and less effluent loads, which further confirmed the potential candidacy of the rSAPB enzyme for application in the leather industry to attain an ecofriendly process of animal hide depilation. More interestingly, the findings on the substrate specificity and kinetic properties of the enzyme using the synthetic peptide para-nitroanilide revealed strong preferences for an aliphatic amino-acid (valine at position P1 for keratinases and an aromatic amino-acid (phenylalanine at positions P1/P4 for subtilisins. Molecular modeling suggested the potential involvement of a Leu31 residue in a network of hydrophobic interactions, which could have shaped the S4 substrate binding site. The latter could be enlarged by mutating L31I, fitting more easily in position P4 than a phenylalanine residue. The molecular modeling of SAPB-T33S showed a potential S2 subside widening by a T33S mutation, thus suggesting its importance in substrate specificity.

  20. Probing the crucial role of Leu31 and Thr33 of the Bacillus pumilus CBS alkaline protease in substrate recognition and enzymatic depilation of animal hide.

    Science.gov (United States)

    Zaraî Jaouadi, Nadia; Jaouadi, Bassem; Ben Hlima, Hajer; Rekik, Hatem; Belhoul, Mouna; Hmidi, Maher; Ben Aicha, Houda Slimene; Hila, Chiraz Gorgi; Toumi, Abdessatar; Aghajari, Nushin; Bejar, Samir

    2014-01-01

    The sapB gene, encoding Bacillus pumilus CBS protease, and seven mutated genes (sapB-L31I, sapB-T33S, sapB-N99Y, sapB-L31I/T33S, sapB-L31I/N99Y, sapB-T33S/N99Y, and sapB-L31I/T33S/N99Y) were overexpressed in protease-deficient Bacillus subtilis DB430 and purified to homogeneity. SAPB-N99Y and rSAPB displayed the highest levels of keratinolytic activity, hydrolysis efficiency, and enzymatic depilation. Interestingly, and at the semi-industrial scale, rSAPB efficiently removed the hair of goat hides within a short time interval of 8 h, thus offering a promising opportunity for the attainment of a lime and sulphide-free depilation process. The efficacy of the process was supported by submitting depilated pelts and dyed crusts to scanning electron microscopic analysis, and the results showed well opened fibre bundles and no apparent damage to the collagen layer. The findings also revealed better physico-chemical properties and less effluent loads, which further confirmed the potential candidacy of the rSAPB enzyme for application in the leather industry to attain an ecofriendly process of animal hide depilation. More interestingly, the findings on the substrate specificity and kinetic properties of the enzyme using the synthetic peptide para-nitroanilide revealed strong preferences for an aliphatic amino-acid (valine) at position P1 for keratinases and an aromatic amino-acid (phenylalanine) at positions P1/P4 for subtilisins. Molecular modeling suggested the potential involvement of a Leu31 residue in a network of hydrophobic interactions, which could have shaped the S4 substrate binding site. The latter could be enlarged by mutating L31I, fitting more easily in position P4 than a phenylalanine residue. The molecular modeling of SAPB-T33S showed a potential S2 subside widening by a T33S mutation, thus suggesting its importance in substrate specificity.

  1. High Iridium concentration of alkaline rocks of Deccan and implications to K/T boundary

    Indian Academy of Sciences (India)

    P N Shukla; N Bhandari; Anirban Das; A D Shukla; J S Ray

    2001-06-01

    We report here an unusually high concentration of iridium in some alkali basalts and alkaline rocks of Deccan region having an age of about 65Ma, similar to the age of the Cretaceous-Tertiary boundary. The alkali basalts of Anjar, in the western periphery of Deccan province, have irid-ium concentration as high as 178pg/g whereas the alkaline rocks and basalts associated with the Amba Dongar carbonatite complex have concentrations ranging between 8 and 80 pg/g. Some of these values are more than an order of magnitude higher than the concentration in the tholeiiticbasalts of Deccan, indicating the significance of alkaline magmatism in the iridium inventory at the Cretaceous-Tertiary boundary. Despite higher concentration, their contribution to the global inventory of iridium in the Cretaceous-Tertiary boundary clays remains small. The concentration of iridium in uorites from Amba Dongar was found to be < 30 pg/g indicating that iridium is not incorporated during their formation in hydrothermal activity.

  2. Fungal protease.

    NARCIS (Netherlands)

    Buxton, F.; Jarai, G.; Visser, J.

    1994-01-01

    The present invention concerns a novel DNA sequence coding for an Aspergillus aspartic protease, an Aspergillus aspartic protease per se and a method for the preparation thereof. The invention further concerns a novel Aspergillus mutant strain defective in a protease of the aspartic proteinase-type,

  3. Highly efficient recombinant production and purification of streptococcal cysteine protease streptopain with increased enzymatic activity.

    Science.gov (United States)

    Lane, Michael D; Seelig, Burckhard

    2016-05-01

    Streptococcus pyogenes produces the cysteine protease streptopain (SpeB) as a critical virulence factor for pathogenesis. Despite having first been described seventy years ago, this protease still holds mysteries which are being investigated today. Streptopain can cleave a wide range of human proteins, including immunoglobulins, the complement activation system, chemokines, and structural proteins. Due to the broad activity of streptopain, it has been challenging to elucidate the functional results of its action and precise mechanisms for its contribution to S. pyogenes pathogenesis. To better study streptopain, several expression and purification schemes have been developed. These methods originally involved isolation from S. pyogenes culture but were more recently expanded to include recombinant Escherichia coli expression systems. While substantially easier to implement, the latter recombinant approach can prove challenging to reproduce, often resulting in mostly insoluble protein and poor purification yields. After extensive optimization of a wide range of expression and purification conditions, we applied the autoinduction method of protein expression and developed a two-step column purification scheme that reliably produces large amounts of purified soluble and highly active streptopain. This method reproducibly yielded 3 mg of streptopain from 50 mL of expression culture at >95% purity, with an activity of 5306 ± 315 U/mg, and no remaining affinity tags or artifacts from recombinant expression. This improved method therefore enables the facile production of the important virulence factor streptopain at higher yields, with no purification scars that might bias functional studies, and with an 8.1-fold increased enzymatic activity compared to previously described procedures.

  4. High-rate capability of zinc anodes in alkaline primary cells

    Science.gov (United States)

    Huot, Jean-Yves; Malservisi, Martin

    This work is devoted to the electrochemical aspects of high-power testing of primary alkaline LR6 ("AA") cells and to the factors influencing cell performance, namely the corresponding zinc anode behaviour under such high-rate conditions. The influence of the high-rate testing regime, such as the discharge mode and the end-potential, on zinc utilisation in alkaline cells has been monitored and its behaviour has been isolated by means of a pseudo-reference electrode. As anticipated, anode formulation, including zinc alloy composition and size distribution, is found to affect the cell's discharge curve and the corresponding zinc electrode potential and utilisation. The effects of these parameters on the discharge curve are discussed in terms of three stages of discharge. Finally, the high-rate capability of commercial LR6 cells is analysed in terms of zinc anode formulation. It was concluded that zinc electrode polarisation is very small and is relatively independent of manufacturer, of zinc anode formulation and of zinc alloying. On the other hand, metallic zinc utilisation remains very low under high-rate conditions.

  5. Alkaline electrolysis cell at high temperature and pressure of 250 °C and 42 bar

    DEFF Research Database (Denmark)

    Allebrod, Frank; Chatzichristodoulou, Christodoulos; Mogensen, Mogens Bjerg

    2013-01-01

    A new type of alkaline electrolysis cells with nickel foam based gas diffusion electrodes and KOH (aq) immobilized in mesoporous SrTiO3 has been developed and tested at temperatures and pressures up to 250 °C and 42 bar, respectively. Current densities of 1.0 A cm−2 have been measured at a cell...... voltage of 1.5 V without the use of expensive noble metal catalysts. High electrical efficiency and current density combined with relatively small production costs may lead to both reduced investment and operating costs for hydrogen and oxygen production....

  6. Use of highly alkaline conditions to improve cost-effectiveness of algal biotechnology.

    Science.gov (United States)

    Canon-Rubio, Karen A; Sharp, Christine E; Bergerson, Joule; Strous, Marc; De la Hoz Siegler, Hector

    2016-02-01

    Phototrophic microorganisms have been proposed as an alternative to capture carbon dioxide (CO2) and to produce biofuels and other valuable products. Low CO2 absorption rates, low volumetric productivities, and inefficient downstream processing, however, currently make algal biotechnology highly energy intensive, expensive, and not economically competitive to produce biofuels. This mini-review summarizes advances made regarding the cultivation of phototrophic microorganisms at highly alkaline conditions, as well as other innovations oriented toward reducing the energy input into the cultivation and processing stages. An evaluation, in terms of energy requirements and energy return on energy invested, is performed for an integrated high-pH, high-alkalinity growth process that uses biofilms. Performance in terms of productivity and expected energy return on energy invested is presented for this process and is compared to previously reported life cycle assessments (LCAs) for systems at near-neutral pH. The cultivation of alkaliphilic phototrophic microorganisms in biofilms is shown to have a significant potential to reduce both energy requirements and capital costs.

  7. High-Strength / High Alkaline Resistant Fe-Phosphate Glass Fibers as Concrete Reinforcement

    Energy Technology Data Exchange (ETDEWEB)

    Mariano Velez

    2008-03-31

    Calcium-iron-phosphate glasses were developed whose chemical durabilities in alkaline solutions (pH 13) were comparable or superior to those of commercial alkaline-resistant (AR) silica-based glasses. However, the tensile strength of Ca-Fe-phosphate fibers, after being exposed to alkaline environments, including wet Portland cement pastes, is lower than that of current AR silicate fibers. Another series of Ca-Fe-phosphate glasses were developed with excellent chemical durability in strong acidic solutions (H2SO4, HF), indicating potential applications where silica-based fibers degrade very quickly, including E-glass. The new Ca-Fe-phosphate glasses can be melted and processed 300 to 500°C lower than silica-based glasses. This offers the possibility of manufacturing glass fibers with lower energy costs by 40-60% and the potential to reduce manufacturing waste and lower gas emissions. It was found that Ca-Fe-phosphate melts can be continuously pulled into fibers depending on the slope of the viscosity-temperature curve and with viscosity ~100 poise, using multi-hole Pt/Rh bushings.

  8. A newly high alkaline lipase: an ideal choice for application in detergent formulations

    Directory of Open Access Journals (Sweden)

    Cherif Slim

    2011-11-01

    Full Text Available Abstract Background Bacterial lipases received much attention for their substrate specificity and their ability to function in extreme environments (pH, temperature.... Many staphylococci produced lipases which were released into the culture medium. Reports of thermostable lipases from Staphylococcus sp. and active in alkaline conditions are not previously described. Results A newly soil-isolated Staphylococcus sp. strain ESW secretes an induced lipase in the culture medium. The effects of temperature, pH and various components in a detergent on the activity and stability of Staphylococcus sp. lipase (SL1 were studied in a preliminary evaluation for use in detergent formulation solutions. The enzyme was highly active over a wide range of pH from 9.0 to 13.0, with an optimum at pH 12.0. The relative activity at pH 13.0 was about 60% of that obtained at pH 12.0. It exhibited maximal activity at 60°C. This novel lipase, showed extreme stability towards non-ionic and anionic surfactants after pre-incubation for 1 h at 40°C, and relative stability towards oxidizing agents. Additionally, the crude enzyme showed excellent stability and compatibility with various commercial solid and liquid detergents. Conclusions These properties added to the high activity in high alkaline pH make this novel lipase an ideal choice for application in detergent formulations.

  9. High Serum Alkaline Phosphatase, Hypercalcaemia, Race, and Mortality in South African Maintenance Haemodialysis Patients

    Science.gov (United States)

    Duarte, Raquel; Naicker, Saraladevi

    2017-01-01

    Objective. To determine the association between serum total alkaline phosphatase (TAP) and mortality in African maintenance haemodialysis patients (MHD). Patients and Methods. The study enrolled a total of 213 patients on MHD from two dialysis centers in Johannesburg between January 2009 and March 2016. Patients were categorized into a low TAP group (≤112 U/L) versus a high TAP group (>112 U/L) based on a median TAP of 112 U/L. Results. During the follow-up period of 7 years, there were 55 (25.8%) deaths. After adjusting for cofounders such as age, other markers of bone disorder, and comorbidity (diabetes mellitus), patients in the high TAP group had significantly higher risk of death compared to patients in the low TAP group (hazard ratio, 2.50; 95% CI 1.24–5.01, P = 0.01). Similarly, serum calcium >2.75 mmol/L was associated with increased risk of death compared to patients within levels of 2.10–2.37 mmol/L (HR 6.34, 95% CI 1.40–28.76; P = 0.02). The HR for death in white patients compared to black patients was 6.88; 95% CI 1.82–25.88; P = 0.004. Conclusion. High levels of serum alkaline phosphatase, hypercalcaemia, and white race are associated with increased risk of death in MHD patients. PMID:28168054

  10. Regional scale hydrological and biogeochemical processes controlling high biodiversity of a groundwater fed alkaline fen

    Science.gov (United States)

    van der Zee, Sjoerd E. A. T. M.; (D. G.) Cirkel, Gijsbert; (J. P. M) witte, Flip

    2014-05-01

    The high floral biodiversity of groundwater fed fens and mesotrophic grasslands depends on the different chemical signatures of the shallow rainwater fed topsoil water and the slightly deeper geochemically affected groundwater. The relatively abrupt gradients between these two layers of groundwater enable the close proximity of plants that require quite different site factors and have different rooting depths. However, sulphur inflow into such botanically interesting areas is generally perceived as a major threat to biodiversity. Although in Europe atmospheric deposition of sulphur has decreased considerably over the last decades, groundwater pollution by sulphate may still continue due to pyrite oxidation in soil as a result of excessive fertilisation. Inflowing groundwater rich in sulphate can change biogeochemical cycling in nutrient-poor wetland ecosystems because of 'so called' internal eutrophication as well as the accumulation of dissolved sulphide, which is phytotoxic. Complementary to conventions, we propose that upwelling sulphate rich groundwater may, in fact, promote the conservation of rare and threatened alkaline fens: excessive fertilisation and pyrite oxidation also produces acidity, which invokes calcite dissolution, and increased alkalinity and hardness of the inflowing groundwater. For a very species-rich wetland nature reserve, we show that sulphate is reduced and effectively precipitated as iron sulphides, when this calcareous and sulphate rich groundwater flows upward through the organic soil of the investigated nature reserve. Also, we show that sulphate reduction occurs simultaneously with an increase in alkalinity production, which in our case results in active calcite precipitation in the soil. In spite of the occurring sulphate reduction, we found no evidence for internal eutrophication. Extremely low phosphorous concentration in the pore water could be attributed to a high C:P ratio of soil organic matter and co-precipitation with

  11. 一株产碱性蛋白酶芽孢杆菌的筛选、鉴定及应用初探%Screening, Identification and Application of a New Bacillus Producing Alkaline Protease

    Institute of Scientific and Technical Information of China (English)

    徐雅芫; 李吕木; 许发芝; 丁小玲; 钱坤

    2011-01-01

    To screen a bacterium producing alkaline protease and study its fermenting effect on protein feeds, a new strain was isolated from topsoil surface of meat power plant and purified using skim milk media; its taxonomy was established according to its morphological features, physiological and biochemical characteristics as well as phylogenetic analysis of 16S Rrna gene sequences; effects of inoculation, culture time of seed liquid and solid-state fermentation time on fennented performance of soybean meal and blood powder were investigated by orthogonal experiments. Results showed that this strain was the most closely related to Bacillus subtilis and named Bacillus subtilis strain D-2; under optional fermentation conditions, the contents of small peptides were significantly increased to 17. 06% and 12. 73% in fermented soybean meal and blood powder, respectively; these optional fermenting conditions as follows; the inculum of 6% , seedliquid culturing time of 24 h, and solid-state fermentation time of 48 h. These results indicated that alkaline protease produced by Bacillus subtilis strain D-2 effectively decomposed protein in both soybean meal and blood powder, which improved dietary performance of protein feeds.%为了筛选碱性蛋白酶产生菌并探讨其对蛋白质饲料的发酵效果,以肉粉厂表层土壤为菌株分离源,利用脱脂牛奶培养基分离和纯化蛋白酶产生菌,通过形态特征、生理生化和16S rRNA基因序列分析确定菌株的分类地位,并采用L9(33)正交设计研究筛选出的优势菌种的接种量(3%、6%和12%)、种子液培养时间(12 h、24h和48 h)和固态发酵时间(12 h、24 h和48 h)3因素对发酵豆粕和血粉的影响.此碱性蛋白酶产生菌与枯草芽孢杆菌亲缘关系最近,命名为枯草芽孢杆菌D-2.在最优条件下,该菌固态发酵豆粕及血粉可显著增加小肽含量至17.06%和12.73%;其最优条件为接种量6%、种子液培养时间24 h和发酵时间48

  12. Highly sensitive and adaptable fluorescence-quenched pair discloses the substrate specificity profiles in diverse protease families

    Science.gov (United States)

    Poreba, Marcin; Szalek, Aleksandra; Rut, Wioletta; Kasperkiewicz, Paulina; Rutkowska-Wlodarczyk, Izabela; Snipas, Scott J.; Itoh, Yoshifumi; Turk, Dusan; Turk, Boris; Overall, Christopher M.; Kaczmarek, Leszek; Salvesen, Guy S.; Drag, Marcin

    2017-01-01

    Internally quenched fluorescent (IQF) peptide substrates originating from FRET (Förster Resonance Energy Transfer) are powerful tool for examining the activity and specificity of proteases, and a variety of donor/acceptor pairs are extensively used to design individual substrates and combinatorial libraries. We developed a highly sensitive and adaptable donor/acceptor pair that can be used to investigate the substrate specificity of cysteine proteases, serine proteases and metalloproteinases. This novel pair comprises 7-amino-4-carbamoylmethylcoumarin (ACC) as the fluorophore and 2,4-dinitrophenyl-lysine (Lys(DNP)) as the quencher. Using caspase-3, caspase-7, caspase-8, neutrophil elastase, legumain, and two matrix metalloproteinases (MMP2 and MMP9), we demonstrated that substrates containing ACC/Lys(DNP) exhibit 7 to 10 times higher sensitivity than conventional 7-methoxy-coumarin-4-yl acetic acid (MCA)/Lys(DNP) substrates; thus, substantially lower amounts of substrate and enzyme can be used for each assay. We therefore propose that the ACC/Lys(DNP) pair can be considered a novel and sensitive scaffold for designing substrates for any group of endopeptidases. We further demonstrate that IQF substrates containing unnatural amino acids can be used to investigate protease activities/specificities for peptides containing post-translationally modified amino acids. Finally, we used IQF substrates to re-investigate the P1-Asp characteristic of caspases, thus demonstrating that some human caspases can also hydrolyze substrates after glutamic acid. PMID:28230157

  13. High Temperature and Pressure Alkaline Electrochemical Reactor for Conversion of Power to Chemicals

    DEFF Research Database (Denmark)

    Chatzichristodoulou, Christodoulos

    2016-01-01

    Moving away from fossil fuels requires harvesting more and more intermittent renewable energy resources and establishing a sustainable system for the production of chemicals. This brings forward the need for efficient large scale energy storage technologies 1-3 and technologies for the conversion...... densities. This work will provide an overview of our efforts to develop components of such high temperature alkaline electrochemical reactors for different applications. Low-cost large-scale production methods have been successfully employed for the production of ceramic diaphragms and full cells...... of renewable electricity to chemicals. Electrochemical reactors can play a crucial role in this endeavor, since they can efficiently and reversibly transform electricity to high-value chemicals, and thus serve as energy storage and recovery devices for balancing the grid, while offering a means...

  14. Influence of ensiling, exogenous protease addition, and bacterial inoculation on fermentation profile, nitrogen fractions, and ruminal in vitro starch digestibility in rehydrated and high-moisture corn.

    Science.gov (United States)

    Ferraretto, L F; Fredin, S M; Shaver, R D

    2015-10-01

    Exogenous protease addition may be an option to increase proteolysis of zein proteins and thus starch digestibility in rehydrated and high-moisture corn (HMC) ensiled for short periods. In addition, microbial inoculation may accelerate fermentation and increase acid production and thus increase solubilization of zein proteins. Four experiments were performed to evaluate the effect on fermentation profile, N fractions, and ruminal in vitro starch digestibility (ivSD) of the following: (1) rehydration and ensiling of dry ground corn; (2) exogenous protease addition to rehydrated un-ensiled and ensiled corn; (3) exogenous protease addition or inoculation in rehydrated ensiled corn; and (4) exogenous protease addition or inoculation in HMC. Experiments 1, 2, and 3 were performed with 7 treatments: dry ground corn (DGC); DGC rehydrated to a targeted dry matter content of 70% (REH); REH treated with exogenous protease (REH+); REH ensiled for 30 d (ENS); ENS treated with exogenous protease (ENS+); ENS treated with a microbial inoculant containing Lactobacillus plantarum, Lactobacillus casei, Enterococcus faecium, and Pediococcus sp. (ENSI); and ENS treated with exogenous protease and microbial inoculant (ENSI+). Experiment 1 compared DGC, REH, and ENS with ivSD being greater for ENS (64.9%) than DGC and REH (51.7% on average). Experiment 2 compared REH and ENS without or with exogenous protease addition (REH+ and ENS+, respectively). Ensiling and exogenous protease addition increased ivSD, but exogenous protease addition was more effective in ENS than REH (6.4 vs. 2.6 percentage unit increase). Experiment 3 compared the effects of exogenous protease addition and inoculation in ENS corn (ENS, ENS+, ENSI, and ENSI+). The addition of protease, but not inoculant, increased ivSD. Inoculation reduced pH and acetate, propionate, and ethanol concentrations, and increased lactate and total acid concentrations. In experiment 4, 8 treatments were a combination of HMC noninoculated

  15. Trichoderma harzianum transformant has high extracellular alkaline proteinase expression during specific mycoparasitic interactions

    Directory of Open Access Journals (Sweden)

    Goldman Maria Helena S.

    1998-01-01

    Full Text Available The mycoparasite Trichoderma harzianum produces an alkaline proteinase that may be specifically involved in mycoparasitism. We have constructed transformant strains of this fungus that overexpress this alkaline proteinase. Some of the transformants were assessed for alkaline proteinase activity, and those with higher activity than the wild type were selected for further studies. One of these transformant strains produced an elevated and constitutive pbr1 mRNA level during mycoparasitic interactions with Rhizoctonia solani.

  16. Protease inhibitor

    DEFF Research Database (Denmark)

    2009-01-01

    The present invention relates to a polypeptide exhibiting a protease inhibitory activity and uses of said polypeptide in methods for inhibiting, directly or indirectly, one or more proteases of the blood clotting cascade. The invention also relates to use of said polypeptide as a pharmaceutical e...

  17. Thermal poling of alkaline earth boroaluminosilicate glasses with intrinsically high dielectric breakdown strength

    Science.gov (United States)

    Smith, Nicholas J.; Lanagan, Michael T.; Pantano, Carlo G.

    2012-04-01

    Per the rectification model of thermal poling, it has been proposed that intrinsic breakdown strength plays a strong limiting role in the internal DC fields supported by the glass from the poling process. One might therefore hypothesize proportionately larger second-order nonlinearity (SON) in glasses with intrinsically high dielectric breakdown strength. We test these ideas by thermal poling of two different commercial alkali-free alkaline-earth boroaluminosilicate display glasses—one with barium only (AF45 from Schott), and the other with a mixture of alkaline-earth ions (OA-10 G from NEG). Not only are such compositions relevant from a commercial standpoint, they are also interesting in that they have been recently shown to exhibit remarkably high intrinsic dielectric breakdown strengths of 11-14 MV/cm. Quantitative Maker fringe and stack Maker-fringe measurements provide an accurate evaluation of the poling-induced SON susceptibilities, and indicate maximum χ(2) values of 0.44 and 0.26 pm/V in these glasses. These values are comparable to those reported for silica and other multicomponent glasses. Thus, the hypothesis that higher χ(2) would be observed in high intrinsic breakdown strength glasses was not validated. Based on our application of the rectification model, internal fields of the order 2-4 MV/cm were calculated, which are well below the measured intrinsic breakdown strengths at room temperature. The most plausible explanation for these observations is nonlinear electronic conduction effects taking place within the depletion region at the poling temperature, limiting internal fields to a fraction of the breakdown field.

  18. High expression of testes-specific protease 50 is associated with poor prognosis in colorectal carcinoma.

    Directory of Open Access Journals (Sweden)

    Lei Zheng

    Full Text Available BACKGROUND: Testes-specific protease 50 (TSP50 is normally expressed in testes and abnormally expressed in breast cancer, but whether TSP50 is expressed in colorectal carcinoma (CRC and its clinical significance is unclear. We aimed to detect TSP50 expression in CRC, correlate it with clinicopathological factors, and assess its potential diagnostic and prognostic value. METHODOLOGY/PRINCIPAL FINDINGS: TSP50 mRNAs and proteins were detected in 7 CRC cell lines and 8 CRC specimens via RT-PCR and Western blot analysis. Immunohistochemical analysis of TSP50, p53 and carcinoembryonic antigen (CEA with tissue microarrays composed of 95 CRCs, 20 colorectal adenomas and 20 normal colorectal tissues were carried out and correlated with clinicopathological characteristics and disease-specific survival for CRC patients. There was no significant correlation between the expression levels of TSP50 and p53 (P = 0.751 or CEA (P = 0.663. Abundant expression of TSP50 protein was found in CRCs (68.4% while it was poorly expressed in colorectal adenomas and normal tissues (P<0.0001. Thus, CRCs can be distinguished from them with high specificity (92.5% and positive predictive value (PPV, 95.6%. The survival of CRC patients with high TSP50 expression was significantly shorter than that of the patients with low TSP50 expression (P = 0.010, specifically in patients who had early-stage tumors (stage I and II; P = 0.004. Multivariate Cox regression analysis indicated that high TSP50 expression was a statistically significant independent risk factor (hazard ratio  = 2.205, 95% CI = 1.214-4.004, P = 0.009. CONCLUSION: Our data demonstrate that TSP50 is a potential effective indicator of poor survival for CRC patients, especially for those with early-stage tumors.

  19. High-Efficiency Artificial Photosynthesis Using a Novel Alkaline Membrane Cell

    Science.gov (United States)

    Narayan, Sri; Haines, Brennan; Blosiu, Julian; Marzwell, Neville

    2009-01-01

    A new cell designed to mimic the photosynthetic processes of plants to convert carbon dioxide into carbonaceous products and oxygen at high efficiency, has an improved configuration using a polymer membrane electrolyte and an alkaline medium. This increases efficiency of the artificial photosynthetic process, achieves high conversion rates, permits the use of inexpensive catalysts, and widens the range of products generated by this type of process. The alkaline membrane electrolyte allows for the continuous generation of sodium formate without the need for any additional separation system. The electrolyte type, pH, electrocatalyst type, and cell voltage were found to have a strong effect on the efficiency of conversion of carbon dioxide to formate. Indium electrodes were found to have higher conversion efficiency compared to lead. Bicarbonate electrolyte offers higher conversion efficiency and higher rates than water solutions saturated with carbon dioxide. pH values between 8 and 9 lead to the maximum values of efficiency. The operating cell voltage of 2.5 V, or higher, ensures conversion of the carbon dioxide to formate, although the hydrogen evolution reaction begins to compete strongly with the formate production reaction at higher cell voltages. Formate is produced at indium and lead electrodes at a conversion efficiency of 48 mg of CO2/kilojoule of energy input. This efficiency is about eight times that of natural photosynthesis in green plants. The electrochemical method of artificial photosynthesis is a promising approach for the conversion, separation and sequestration of carbon dioxide for confined environments as in space habitats, and also for carbon dioxide management in the terrestrial context. The heart of the reactor is a membrane cell fabricated from an alkaline polymer electrolyte membrane and catalyst- coated electrodes. This cell is assembled and held in compression in gold-plated hardware. The cathode side of the cell is supplied with carbon

  20. Highly efficient synthesis of endomorphin-2 under thermodynamic control catalyzed by organic solvent stable proteases with in situ product removal.

    Science.gov (United States)

    Xu, Jiaxing; Sun, Honglin; He, Xuejun; Bai, Zhongzhong; He, Bingfang

    2013-02-01

    An efficient enzymatic synthesis of endomorphin-2 (EM-2) was achieved using organic solvent stable proteases in nonaqeous media, based on thermodynamic control and an in situ product removal methodology. The high stability of biocatalysts in organic solvents enabled the aleatoric modulation of the nonaqueous reaction media to shift thermodynamic equilibrium toward synthesis. Peptide Boc-Phe-Phe-NH2 was synthesized with a high yield of 96% by the solvent stable protease WQ9-2 in monophase medium with an economical molar ratio of the substrate of 1:1. The tetrapeptide Boc-Tyr-Pro-Phe-Phe-NH2 was synthesized with a yield of 88% by another organic solvent tolerant protease PT121 from Boc-Tyr-Pro-OH and Phe-Phe-NH2 in an organic-aqueous biphasic system. The reaction-separation coupling in both enzymatic processes provides "driving forces" for the synthetic reactions and gives a high yield and high productivity without purification of the intermediate, thereby making the synthesis more amenable to scale-up. Copyright © 2012 Elsevier Ltd. All rights reserved.

  1. Highly efficient anode catalyst with a Ni@PdPt core–shell nanostructure for methanol electrooxidation in alkaline media

    Institute of Scientific and Technical Information of China (English)

    Pei-shu Yu; Chun-tao Liu; Bo Feng; Jia-feng Wan; Li Li; Chun-yu Du

    2015-01-01

    To enhance the electrocatalytic activity of anode catalysts used in alkaline-media direct methanol fuel cells (DMFCs), a Ni@PdPt electrocatalyst was successfully prepared using a three-phase-transfer method. The Ni@PdPt electrocatalyst was characterized by X-ray dif-fraction (XRD), X-ray photoelectron spectroscopy (XPS), transmission electron microscopy (TEM), and high-resolution TEM (HRTEM) techniques. The experimental results indicate that the average particle size of the core–shell-structured Ni@PdPt electrocatalyst is approxi-mately 5.6 nm. The Ni@PdPt electrocatalyst exhibits a catalytic activity 3.36 times greater than that of PdPt alloys for methanol oxidation in alkaline media. The developed Ni@PdPt electrocatalyst offers a promising alternative as a highly electrocatalytically active anode catalyst for alkaline DMFCs.

  2. tolerant alkaline protease from Bacillus coagulans PSB

    African Journals Online (AJOL)

    oyaide

    2013-05-22

    May 22, 2013 ... optimum activity at 60°C and pH 8.0 with casein as substrate. The enzyme was .... Hydrolysis of protein substrates such as casein, bovine serum albumin (BSA) .... the enhancement of hydrophobic interactions inside protein ...

  3. Modulation of the epithelial sodium channel (ENaC) by bacterial metalloproteases and protease inhibitors.

    Science.gov (United States)

    Butterworth, Michael B; Zhang, Liang; Liu, Xiaoning; Shanks, Robert M; Thibodeau, Patrick H

    2014-01-01

    The serralysin family of metalloproteases is associated with the virulence of multiple gram-negative human pathogens, including Pseudomonas aeruginosa and Serratia marcescens. The serralysin proteases share highly conserved catalytic domains and show evolutionary similarity to the mammalian matrix metalloproteases. Our previous studies demonstrated that alkaline protease (AP) from Pseudomonas aeruginosa is capable of activating the epithelial sodium channel (ENaC), leading to an increase in sodium absorption in airway epithelia. The serralysin proteases are often co-expressed with endogenous, intracellular or periplasmic inhibitors, which putatively protect the bacterium from unwanted or unregulated protease activities. To evaluate the potential use of these small protein inhibitors in regulating the serralysin induced activation of ENaC, proteases from Pseudomonas aeruginosa and Serratia marcescens were purified for characterization along with a high affinity inhibitor from Pseudomonas. Both proteases showed activity against in vitro substrates and could be blocked by near stoichiometric concentrations of the inhibitor. In addition, both proteases were capable of activating ENaC when added to the apical surfaces of multiple epithelial cells with similar slow activation kinetics. The high-affinity periplasmic inhibitor from Pseudomonas effectively blocked this activation. These data suggest that multiple metalloproteases are capable of activating ENaC. Further, the endogenous, periplasmic bacterial inhibitors may be useful for modulating the downstream effects of the serralysin virulence factors under physiological conditions.

  4. Modulation of the epithelial sodium channel (ENaC by bacterial metalloproteases and protease inhibitors.

    Directory of Open Access Journals (Sweden)

    Michael B Butterworth

    Full Text Available The serralysin family of metalloproteases is associated with the virulence of multiple gram-negative human pathogens, including Pseudomonas aeruginosa and Serratia marcescens. The serralysin proteases share highly conserved catalytic domains and show evolutionary similarity to the mammalian matrix metalloproteases. Our previous studies demonstrated that alkaline protease (AP from Pseudomonas aeruginosa is capable of activating the epithelial sodium channel (ENaC, leading to an increase in sodium absorption in airway epithelia. The serralysin proteases are often co-expressed with endogenous, intracellular or periplasmic inhibitors, which putatively protect the bacterium from unwanted or unregulated protease activities. To evaluate the potential use of these small protein inhibitors in regulating the serralysin induced activation of ENaC, proteases from Pseudomonas aeruginosa and Serratia marcescens were purified for characterization along with a high affinity inhibitor from Pseudomonas. Both proteases showed activity against in vitro substrates and could be blocked by near stoichiometric concentrations of the inhibitor. In addition, both proteases were capable of activating ENaC when added to the apical surfaces of multiple epithelial cells with similar slow activation kinetics. The high-affinity periplasmic inhibitor from Pseudomonas effectively blocked this activation. These data suggest that multiple metalloproteases are capable of activating ENaC. Further, the endogenous, periplasmic bacterial inhibitors may be useful for modulating the downstream effects of the serralysin virulence factors under physiological conditions.

  5. High Variability of Plasma Drug Concentrations in Dual Protease Inhibitor Regimens

    Science.gov (United States)

    Guiard-Schmid, Jean-Baptiste; Poirier, Jean-Marie; Meynard, Jean-Luc; Bonnard, Philippe; Gbadoe, Ayi Hola; Amiel, Corinne; Calligaris, Frédérique; Abraham, Bruno; Pialoux, Gilles; Girard, Pierre-Marie; Jaillon, Patrice; Rozenbaum, Willy

    2003-01-01

    Ritonavir (RTV) strongly increases the concentrations of protease inhibitors (PIs) in plasma in patients given a combination of RTV and another PI. This pharmacological interaction is complex and poorly characterized and shows marked inter- and intraindividual variations. In addition, RTV interacts differently with saquinavir (SQV), indinavir (IDV), amprenavir (APV), and lopinavir (LPV). In this retrospective study on 542 human immunodeficiency virus-infected patients, we compared inter- and intraindividual variability of plasma PI concentrations and correlations between the Cmin (minimum concentration of drug in plasma) values for RTV and the coadministered PI Cmin values. Mean RTV Cmins are significantly lower in patients receiving combinations containing APV or LPV than in combinations with SQV or IDV. With the most common PI dose regimens (600 mg of IDV twice a day [BID], 800 mg of SQV BID, and 400 mg of LPV BID), the interindividual Cmin variability of patients treated with a PI and RTV seemed to be lower with APV and LPV than with IDV and SQV. As regards intraindividual variability, APV also differed from the other PIs, exhibiting lower Cmin variability than with the other combinations. Significant positive correlations between RTV Cmin and boosted PI Cmin were observed with IDV, SQV, and LPV, but not with APV. Individual dose adjustments must take into account the specificity the pharmacological interaction of each RTV/PI combination and the large inter- and intraindividual variability of plasma PI levels to avoid suboptimal plasma drug concentrations which may lead to treatment failure and too high concentrations which may induce toxicity and therefore reduce patient compliance. PMID:12604531

  6. Bone mineral density changes in protease inhibitor-sparing vs. nucleoside reverse transcriptase inhibitor-sparing highly active antiretroviral therapy: data from a randomized trial

    DEFF Research Database (Denmark)

    Hansen, Ann-Brit Eg; Obel, N; Nielsen, H

    2011-01-01

    The aim of the study was to compare changes in bone mineral density (BMD) over 144 weeks in HIV-infected patients initiating nucleoside reverse transcriptase inhibitor (NRTI)-sparing or protease inhibitor-sparing highly active antiretroviral therapy (HAART).......The aim of the study was to compare changes in bone mineral density (BMD) over 144 weeks in HIV-infected patients initiating nucleoside reverse transcriptase inhibitor (NRTI)-sparing or protease inhibitor-sparing highly active antiretroviral therapy (HAART)....

  7. Supermarket Proteases.

    Science.gov (United States)

    Hagar, William G.; Bullerwell, Lornie D.

    2003-01-01

    Presents a laboratory activity on enzymes. Uses common items found in the supermarket that contain protease enzymes, such as contact lens cleaner and meat tenderizer. Demonstrates the digestion of gelatin proteins as part of enzymatic reactions. (Author/SOE)

  8. Highly Constrained Bicyclic Scaffolds for the Discovery of Protease-Stable Peptides via mRNA Display.

    Science.gov (United States)

    Hacker, David E; Hoinka, Jan; Iqbal, Emil S; Przytycka, Teresa M; Hartman, Matthew C T

    2017-02-01

    Highly constrained peptides such as the knotted peptide natural products are promising medicinal agents because of their impressive biostability and potent activity. Yet, libraries of highly constrained peptides are challenging to prepare. Here, we present a method which utilizes two robust, orthogonal chemical steps to create highly constrained bicyclic peptide libraries. This technology was optimized to be compatible with in vitro selections by mRNA display. We performed side-by-side monocyclic and bicyclic selections against a model protein (streptavidin). Both selections resulted in peptides with mid-nanomolar affinity, and the bicyclic selection yielded a peptide with remarkable protease resistance.

  9. Alkaline earth silicate wools - A new generation of high temperature insulation.

    Science.gov (United States)

    Brown, Robert C; Harrison, Paul T C

    2012-11-01

    Intensive study of the natural asbestiform minerals that cause human diseases, and the consequent understanding of their hazardous characteristics, has enabled the development of manufactured fibres whose physical and/or chemical properties, in particular as they relate to biopersistence, have been adjusted to minimize possible harm to health. A strong driver for the developmentof new high temperature insulation materials wasthe perception of the toxicity of refractory ceramic fibres (RCF)and their classification in the EU as a category 2 carcinogen under Directive 67/548/EEC. Such classification carries with it the requirement for substitution by less hazardous materials. This paper focuses on the development of alkaline earth silicate (AES) wools as a new class of high temperature insulation with the capability of such substitution in a number of applications. These wools have only a low potential to cause harm because they do not persist in lung tissue once deposited, and have produced minimal effects in experimental test systems. AES wools are increasingly being used in a wide range of high temperature applications.

  10. Biohydrogen Fermentation from Sucrose and Piggery Waste with High Levels of Bicarbonate Alkalinity

    Directory of Open Access Journals (Sweden)

    Jeongdong Choi

    2015-03-01

    Full Text Available This study examined the influence of biohydrogen fermentation under the high bicarbonate alkalinity (BA and pH to optimize these critical parameters. When sucrose was used as a substrate, hydrogen was produced over a wide range of pH values (5–9 under no BA supplementation; however, BA affected hydrogen yield significantly under different initial pHs (5–10. The actual effect of high BA using raw piggery waste (pH 8.7 and BA 8.9 g CaCO3/L showed no biogas production or propionate/acetate accumulation. The maximum hydrogen production rate (0.32 L H2/g volatile suspended solids (VSS-d was observed at pH 8.95 and 3.18 g CaCO3/L. BA greater than 4 g CaCO3/L also triggered lactate-type fermentation, leading to propionate accumulation, butyrate reduction and homoacetogenesis, potentially halting the hydrogen production rate. These results highlight that the substrate with high BA need to amend adequately to maximize hydrogen production.

  11. Characterization of high temperature-tolerant rhizobia isolated from Prosopis juliflora grown in alkaline soil.

    Science.gov (United States)

    Kulkarni, Suneeta; Nautiyal, Chandra Shekhar

    1999-10-01

    A method was developed for the fast screening and selection of high-temperature tolerant rhizobial strains from root nodules of Prosopis juliflora growing in alkaline soils. The high-temperature tolerant rhizobia were selected from 2,500 Rhizobium isolates with similar growth patterns on yeast mannitol agar plates after 72 h incubation at 30 and 45 degrees C, followed by a second screening at 47.5 degrees C. Seventeen high-temperature tolerant rhizobial strains having distinguishable protein band patterns were finally selected for further screening by subjecting them to temperature stress up to 60 degrees C in yeast mannitol broth for 6 h. The high-temperature tolerant strains were NBRI12, NBRI329, NBRI330, NBRI332, and NBRI133. Using this procedure, a large number of rhizobia from root nodules of P. juliflora were screened for high-temperature tolerance. The assimilation of several carbon sources, tolerance to high pH and salt stress, and ability to nodulate P. juliflora growing in a glasshouse and nursery of the strains were studied. All five isolates had higher plant dry weight in the range of 29.9 to 88.6% in comparison with uninoculated nursery-grown plants. It was demonstrated that it is possible to screen in nature for superior rhizobia exemplified by the isolation of temperature-tolerant strains, which established effective symbiosis with nursery-grown P. juliflora. These findings indicate a correlation between strain performance under in vitro stress in pure culture and strain behavior under symbiotic conditions. Pure culture evaluation may be a useful tool in search for Rhizobium strains better suited for soil environments where high temperature, pH, and salt stress constitutes a limitation for symbiotic biological nitrogen fixation.

  12. Anti-obesity effect of alkaline reduced water in high fat-fed obese mice.

    Science.gov (United States)

    Ignacio, Rosa Mistica Coles; Kang, Tae-Young; Kim, Cheol-Su; Kim, Soo-Ki; Yang, Young-Chul; Sohn, Joon-Hyung; Lee, Kyu-Jae

    2013-01-01

    Whether or not alkaline reduced water (ARW) has a positive effect on obesity is unclear. This study aims to prove the positive effect of ARW in high-fat (HF) diet-induced obesity (DIO) in C57BL/6 mice model. Toward this, obesity was induced by feeding the C57BL/6 male mice with high-fat diet (w/w 45% fat) for 12 weeks. Thereafter, the animals were administered with either ARW or tap water. Next, the degree of adiposity and DIO-associated parameters were assessed: clinico-pathological parameters, biochemical measurements, histopathological analysis of liver, the expression of cholesterol metabolism-related genes in the liver, and serum levels of adipokine and cytokine. We found that ARW-fed mice significantly ameliorated adiposity: controlled body weight gain, reduced the accumulation of epididymal fats and decreased liver fats as compared to control mice. Accordingly, ARW coordinated the level of adiponectin and leptin. Further, mRNA expression of cytochrome P450 (CYP)7A1 was upregulated. In summary, our data shows that ARW intake inhibits the progression of HF-DIO in mice. This is the first note on anti-obesity effect of ARW, clinically implying the safer fluid remedy for obesity control.

  13. Enhanced coagulation for high alkalinity and micro-polluted water: the third way through coagulant optimization.

    Science.gov (United States)

    Yan, Mingquan; Wang, Dongsheng; Qu, Jiuhui; Ni, Jinren; Chow, Christopher W K

    2008-04-01

    Conventional coagulation is not an effective treatment option to remove natural organic matter (NOM) in water with high alkalinity/pH. For this type of water, enhanced coagulation is currently proposed as one of the available treatment options and is implemented by acidifying the raw water and applying increased doses of hydrolyzing coagulants. Both of these methods have some disadvantages such as increasing the corrosive tendency of water and increasing cost of treatment. In this paper, an improved version of enhanced coagulation through coagulant optimization to treat this kind of water is demonstrated. A novel coagulant, a composite polyaluminum chloride (HPAC), was developed with both the advantages of polyaluminum chloride (PACl) and the additive coagulant aids: PACl contains significant amounts of highly charged and stable polynuclear aluminum hydrolysis products, which is less affected by the pH of the raw water than traditional coagulants (alum and ferric salts); the additives can enhance both the charge neutralization and bridging abilities of PACl. HPAC exhibited 30% more efficiency than alum and ferric salts in dissolved organic carbon (DOC) removal and was very effective in turbidity removal. This result was confirmed by pilot-scale testing, where particles and organic matter were removed synergistically with HPAC as coagulant by sequential water treatment steps including pre-ozonation, coagulation, flotation and sand filtration.

  14. The Effects of High Alkaline Fly Ash on Strength Behaviour of a Cohesive Soil

    Directory of Open Access Journals (Sweden)

    A. Binal

    2016-01-01

    Full Text Available Contemporarily, there are 16 coal-burning thermal power plants currently operating in Turkey. This number is expected to rise to 46 in the future. Annually, about 15 million tons of fly ash are removed from the existing thermal power plants in Turkey, but a small proportion of it, 2%, is recyclable. Turkey’s plants are fired by lignite, producing Class C fly ash containing a high percentage of lime. Sulfate and alkali levels are also higher in Class C fly ashes. Therefore, fly ash is, commonly, unsuitable as an additive in cement or concrete in Turkey. In this study, highly alkaline fly ash obtained from the Yeniköy thermal power plants is combined with soil samples in different proportions (5%, 10%, 15%, 20%, and 25% and changes in the geomechanical properties of Ankara clay were investigated. The effect of curing time on the physicomechanical properties of the fly ash mixed soil samples was also analyzed. The soil classification of Ankara clay changed from CH to MH due to fly ash additives. Free swelling index values showed a decrease of 92.6%. Direct shear tests on the cohesion value of Ankara clay have shown increases by multiples of 15.85 and 3.01 in internal friction angle values. The California bearing ratio has seen a more drastic increase in value (68.7 times for 25% fly ash mix.

  15. High Variability of Plasma Drug Concentrations in Dual Protease Inhibitor Regimens

    OpenAIRE

    Guiard-Schmid, Jean-Baptiste; Poirier, Jean-Marie; Meynard, Jean-Luc; Bonnard, Philippe; Gbadoe, Ayi Hola; Amiel, Corinne; Calligaris, Frédérique; Abraham, Bruno; Pialoux, Gilles; Girard, Pierre-Marie; Jaillon, Patrice; Rozenbaum, Willy

    2003-01-01

    Ritonavir (RTV) strongly increases the concentrations of protease inhibitors (PIs) in plasma in patients given a combination of RTV and another PI. This pharmacological interaction is complex and poorly characterized and shows marked inter- and intraindividual variations. In addition, RTV interacts differently with saquinavir (SQV), indinavir (IDV), amprenavir (APV), and lopinavir (LPV). In this retrospective study on 542 human immunodeficiency virus-infected patients, we compared inter- and ...

  16. Assessment of commercially available ion exchange materials for cesium removal from highly alkaline wastes

    Energy Technology Data Exchange (ETDEWEB)

    Brooks, K.P.; Kim, A.Y.; Kurath, D.E.

    1996-04-01

    Approximately 61 million gallons of nuclear waste generated in plutonium production, radionuclide removal campaigns, and research and development activities is stored on the Department of Energy`s Hanford Site, near Richland, Washington. Although the pretreatment process and disposal requirements are still being defined, most pretreatment scenarios include removal of cesium from the aqueous streams. In many cases, after cesium is removed, the dissolved salt cakes and supernates can be disposed of as LLW. Ion exchange has been a leading candidate for this separation. Ion exchange systems have the advantage of simplicity of equipment and operation and provide many theoretical stages in a small space. The organic ion exchange material Duolite{trademark} CS-100 has been selected as the baseline exchanger for conceptual design of the Initial Pretreatment Module (IPM). Use of CS-100 was chosen because it is considered a conservative, technologically feasible approach. During FY 96, final resin down-selection will occur for IPM Title 1 design. Alternate ion exchange materials for cesium exchange will be considered at that time. The purpose of this report is to conduct a search for commercially available ion exchange materials which could potentially replace CS-100. This report will provide where possible a comparison of these resin in their ability to remove low concentrations of cesium from highly alkaline solutions. Materials which show promise can be studied further, while less encouraging resins can be eliminated from consideration.

  17. The use of an economical medium for the production of alkaline ...

    African Journals Online (AJOL)

    STORAGESEVER

    2010-05-03

    May 3, 2010 ... marine-processing by-products for the production of alkaline proteases by Bacillus ... Key words: Fish powder, hulled grain of wheat, heads and viscera, alkaline ... detergent industry, because the pH of laundry detergents.

  18. Protease-mediated drug delivery

    Science.gov (United States)

    Dickson, Eva F.; Goyan, Rebecca L.; Kennedy, James C.; Mackay, M.; Mendes, M. A. K.; Pottier, Roy H.

    2003-12-01

    Drugs used in disease treatment can cause damage to both malignant and normal tissue. This toxicity limits the maximum therapeutic dose. Drug targeting is of high interest to increase the therapeutic efficacy of the drug without increasing systemic toxicity. Certain tissue abnormalities, disease processes, cancers, and infections are characterized by high levels of activity of specific extracellular and/or intracellular proteases. Abnormally high activity levels of specific proteases are present at sites of physical or chemical trauma, blood clots, malignant tumors, rheumatoid arthritis, inflammatory bowel disease, gingival disease, glomerulonerphritis, and acute pancreatitis. Abnormal protease activity is suspected in development of liver thrombosis, pulmonary emphysema, atherosclerosis, and muscular dystrophy. Inactiviating disease-associated proteases by the administration of appropriate protease inhibitors has had limited success. Instead, one could use such proteases to target drugs to treat the condition. Protease mediated drug delivery offers such a possibility. Solubilizing groups are attached to insoluble drugs via a polypeptide chain which is specifically cleavable by certian proteases. When the solubilized drug enounters the protease, the solubilizing moieties are cleaved, and the drug precipitates at the disease location. Thus, a smaller systemic dosage could result in a therapeutic drug concentration at the treatment site with less systemic toxicity.

  19. Phosphorylation regulates proteolytic efficiency of TEV protease detected by a 5(6)-carboxyfluorescein-pyrene based fluorescent sensor.

    Science.gov (United States)

    He, Yao-Hui; Li, Yan-Mei; Chen, Yong-Xiang

    2016-04-01

    TEV protease is of great importance for in vitro and in vivo site-specific cleavage of proteins. The proteolytic efficiency of TEV protease is often regulated by mutation of the substrate, which is irreversible and hard to be modulated. Herein, a facile and reversible method, based on phosphorylation in the substrate, is developed to regulate the cleavage capability of TEV protease. Phosphorylation at P3 tyrosine hinders the recognition of TEV protease to the substrate by using a robust fluorescent protease sensor. Moreover, the phosphate group can be easily removed by alkaline phosphatases for recovering the proteolytic efficiency of TEV protease. Additionally, 5(6)-carboxyfluorescein and pyrene have been used as high-efficiency mutual fluorophore-quencher pair in the peptide-based protease sensor for the first time, which provides a chance to simultaneously monitor the cleavage process in two respective fluorescence channels. Further studies indicated both dynamic and static components contributing to the mutual quenching system. The phosphorylation-regulated TEV protease proteolysis system can be used in conditional cleavage of protein or peptide tag.

  20. New high-throughput screening protease assay based upon supramolecular self-assembly.

    Energy Technology Data Exchange (ETDEWEB)

    Whitten, David G.; Tang, Yanli; Zhou, Zhijun; Achyuthan, Komandoor E.

    2008-11-01

    We previously demonstrated that the supramolecular self-assembly of cyanines could be useful for developing fluorescent enzymatic assays. We took that concept a step further by synthesizing a covalent adduct of the tetrapeptide Asp-Glu-Val-Asp (DEVD) and a cyanine (DEVD-cyanine). The DEVD-cyanine due to its canonical sequence was recognized and hydrolyzed by the proteases, Caspase-3 and -7 in 96- or 384-microwell plate reactions. The catalytically liberated cyanine self-assembled upon scaffolds of carboxymethylamylose (CMA), carboxymethylcellulose (CMC), or a mixture of CMA and CMC resulting in a J aggregate exhibiting bright fluorescence at a 470 nm emission wavelength (optimum signal/background using excitation wavelengths of 415-440 nm). The fluorescence intensity increased with enzyme and substrate concentrations or reaction time and exhibited classical saturation profiles of a rectangular hyperbola. Saturation of the reaction was at 30 U/mL (1 {micro}g/mL) Caspase-3 and 250 {micro}M DEVD-cyanine. The reaction kinetics was linear between 1 and 20 min and saturated at 60 min. The affinity constant (Km) for DEVD-cyanine was 23 {micro}M, similar to those of previously reported values for other DEVD substrates of Caspase-3. Maximal fluorescence emission was observed by using a mixture of CMA and CMC scaffolds at 65 and 35 {micro}M, respectively. The reaction kinetics of Caspase-7 executed in a 384-well plate was similar to the reaction kinetics of Caspase-3 conducted in a 96-well plate. We believe that this is the first demonstration of a cyanine liberated from a covalent adduct due to protease action, leading to supramolecular self-assembly and the detection of protease activity.

  1. Identification of novel chromone based sulfonamides as highly potent and selective inhibitors of alkaline phosphatases.

    Science.gov (United States)

    al-Rashida, Mariya; Raza, Rabia; Abbas, Ghulam; Shah, Muhammad Shakil; Kostakis, George E; Lecka, Joanna; Sévigny, Jean; Muddassar, Muhammad; Papatriantafyllopoulou, Constantina; Iqbal, Jamshed

    2013-08-01

    A new series of structurally diverse chromone containing sulfonamides has been developed. Crystal structures of three representative compounds (2a, 3a and 4a) in the series are reported. All compounds were screened for their inhibitory potential against alkaline phosphatases (ALPs). Two main classes of ALP isozymes were selected for this study, the tissue non-specific alkaline phosphatase (TNALP) from bovine and porcine source and the tissue-specific intestinal alkaline phosphatases (IALPs) from bovine source. All sulfonamide compounds had a marked preference for IALP (K(i), up to 0.01 ± 0.001 μM) over TNALPs. Kinetics studies of the compounds showed competitive mode of inhibition. Molecular docking studies were carried out in order to characterize the selective inhibition of the compounds. An additional interesting aspect of these chromone sulfonamides is their inhibitory activity against ecto-5'-nucleotidase enzyme. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  2. Engineered single-domain antibodies with high protease resistance and thermal stability.

    Directory of Open Access Journals (Sweden)

    Greg Hussack

    Full Text Available The extreme pH and protease-rich environment of the upper gastrointestinal tract is a major obstacle facing orally-administered protein therapeutics, including antibodies. Through protein engineering, several Clostridium difficile toxin A-specific heavy chain antibody variable domains (V(HHs were expressed with an additional disulfide bond by introducing Ala/Gly54Cys and Ile78Cys mutations. Mutant antibodies were compared to their wild-type counterparts with respect to expression yield, non-aggregation status, affinity for toxin A, circular dichroism (CD structural signatures, thermal stability, protease resistance, and toxin A-neutralizing capacity. The mutant V(HHs were found to be well expressed, although with lower yields compared to wild-type counterparts, were non-aggregating monomers, retained low nM affinity for toxin A, albeit the majority showed somewhat reduced affinity compared to wild-type counterparts, and were capable of in vitro toxin A neutralization in cell-based assays. Far-UV and near-UV CD spectroscopy consistently showed shifts in peak intensity and selective peak minima for wild-type and mutant V(HH pairs; however, the overall CD profile remained very similar. A significant increase in the thermal unfolding midpoint temperature was observed for all mutants at both neutral and acidic pH. Digestion of the V(HHs with the major gastrointestinal proteases, at biologically relevant concentrations, revealed a significant increase in pepsin resistance for all mutants and an increase in chymotrypsin resistance for the majority of mutants. Mutant V(HH trypsin resistance was similar to that of wild-type V(HHs, although the trypsin resistance of one V(HH mutant was significantly reduced. Therefore, the introduction of a second disulfide bond in the hydrophobic core not only increases V(HH thermal stability at neutral pH, as previously shown, but also represents a generic strategy to increase V(HH stability at low pH and impart

  3. Electromembrane recycling of highly mineralized alkaline blowdown water from evaporative water treatment plants at thermal power stations

    Science.gov (United States)

    Chichirova, N. D.; Chichirov, A. A.; Lyapin, A. I.; Minibaev, A. I.; Silov, I. Yu.; Tolmachev, L. I.

    2016-12-01

    Thermal power stations (TPS) are the main source of highly mineralized effluents affecting the environment. An analysis of their water systems demonstrates that alkaline effluents prevail at TPSs. Extraction of an alkali from highly mineralized effluents can make the recycling of effluents economically feasible. A method is proposed of electromembrane recycling of liquid alkaline highly mineralized wastes from TPSs. The process includes electromembrane apparatuses of two types, namely, a diffusion dialysis extractor (DDE) intended for extraction of the alkali from a highly mineralized solution having a complex composition and an electrodialysis concentrator for increasing the concentration of the extracted solution to a value suitable for use in water treatment plants at TPSs. For implementation of the first process (i.e. the extraction of alkali from alkaline-salt solution) various membranes from various manufacturers were studied: CM-PAD and AM-PAD (Ralex, Czechia), MK-40, MA-40, MA-41, MA-414, and MB-2 (OOO OKhK "Shchekinoazot", Russia), AR103-QDF and CR61-CMP (Ionies Inc., USA). The experiments demonstrate that the acceptable degree of separation of the alkali and the salt is achieved in a pair of cation-exchange membranes with the efficiency of separation being higher without an electric field. The highest efficiency was attained with Russian-made membranes (MK-40, OOO OKhK "Shchekinoazot"). A full scale experiment on recycling of highly-mineralized blowdown water from the evaporating water treatment system at the Kazan cogeneration power station No. 3 (TETs-3) was performed in a pilot unit consisting of two electromembrane apparatuses made by UAB "Membraninės Technologijos LT". In the experiments every ton of blowdown water yielded 0.1 t of concentrated alkaline solution with an alkali content of up to 4 wt % and 0.9 t of the softened salt solution suitable for the reuse in the TPS cycle. The power rate is 6 kWh / ton of blowdown water.

  4. Effects of the aspartic protease inhibitor from Lupinus bogotensis seeds on the growth and development of Hypothenemus hampei: an inhibitor showing high homology with storage proteins.

    Science.gov (United States)

    Molina, Diana; Patiño, Luisa; Quintero, Mónica; Cortes, José; Bastos, Sara

    2014-02-01

    The coffee berry borer Hypothenemus hampei is a pest that causes great economic damage to coffee grains worldwide. Because the proteins consumed are digested by aspartic proteases in the insect's midgut, the inhibition of these proteases by transferring a gene encoding an aspartic protease inhibitor from Lupinus bogotensis Benth. to coffee plants could provide a promising strategy to control this pest. Five aspartic protease inhibitors from L. bogotensis (LbAPI) were accordingly purified and characterized. The gene encoding the L. bogotensis aspartic protease inhibitor (LbAPI), with the highest inhibitory activity against H. hampei, was expressed in Escherichia coli and the purified recombinant protein (rLbAPI), with a molecular mass of 15 kDa, was subsequently assessed for its ability to inhibit the aspartic protease activity present in the H. hampei midgut in vitro, as well as its effects on the growth and development of H. hampei in vivo. The in vitro experiments showed that rLbAPI was highly effective against aspartic proteases from H. hampei guts, with a half maximal inhibitory concentration (IC50) of 2.9 μg. The in vivo experiments showed that the concentration of rLbAPI (w/w) in the artificial diet necessary to cause 50% mortality (LD50) of the larvae was 0.91%. The amino acid sequence of LbAPI had high homology (52-80%) to the seed storage proteins, vicilin and β-conglutin, suggesting that this protein was generated by evolutionary events from a β-conglutin precursor. Based on these results, LbAPI may have a dual function as storage protein, and as defense protein against H. hampei. These results provide a promising alternative to obtain a coffee plant resistant to H. hampei. Copyright © 2013 Elsevier Ltd. All rights reserved.

  5. INFLUENCE OF VARIOUS ENVIRONMENTAL PARAMETERS ON PROTEASE SECRETION FROM BACILLUS SUBTILIS DKMNR

    Directory of Open Access Journals (Sweden)

    D. Kezia

    2011-03-01

    Full Text Available A protease producing microorganism was isolated from soil collected from garden soil samples around the department of Chemical Engineering, Andhra University, Vizag, A.P, India and identified as bacillus species. The isolate DKMNR produced alkaline protease the optimum conditions for protease activity was 330C at pH 9 with 2% inoculum in the medium after 24 h of incubation with an agitation speed of 200 rpm and EDTA was found to be inhibitor of alkaline protease. The extracellular production of the enzyme, its alkaline nature and compatibility with most commercial detergents are features which suggest its application in the detergent industry.

  6. Early containment of high-alkaline solution simulating low-level radioactive waste stream in clay-bearing blended cement

    Energy Technology Data Exchange (ETDEWEB)

    Kruger, A.A. [Westinghouse Hanford Co., Richland, WA (United States); Olson, R.A.; Tennis, P.D. [Northwestern Univ., Evanston, IL (United States). Center for Advanced Cement-Based Materials] [and others

    1995-04-01

    Portland cement blended with fly ash and attapulgite clay was mixed with high-alkaline solution simulating low-level radioactive waste stream at a one-to-one weight ratio. Mixtures were adiabatically and isothermally cured at various temperatures and analyzed for phase composition, total alkalinity, pore solution chemistry, and transport properties as measured by impedance spectroscopy. Total alkalinity is characterized by two main drops. The early one corresponds to a rapid removal of phosphorous, aluminum, sodium, and to a lesser extent potassium solution. The second drop from about 10 h to 3 days is mainly associated with the removal of aluminum, silicon, and sodium. Thereafter, the total alkalinity continues descending, but at a lower rate. All pastes display a rapid flow loss that is attributed to an early precipitation of hydrated products. Hemicarbonate appears as early as one hour after mixing and is probably followed by apatite precipitation. However, the former is unstable and decomposes at a rate that is inversely related to the curing temperature. At high temperatures, zeolite appears at about 10 h after mixing. At 30 days, the stabilized crystalline composition Includes zeolite, apatite and other minor amounts of CaCO{sub 3}, quartz, and monosulfate Impedance spectra conform with the chemical and mineralogical data. The normalized conductivity of the pastes shows an early drop, which is followed by a main decrease from about 12 h to three days. At three days, the permeability of the cement-based waste as calculated by Katz-Thompson equation is over three orders of magnitude lower than that of ordinary portland cement paste. However, a further decrease in the calculated permeability is questionable. Chemical stabilization is favorable through incorporation of waste species into apatite and zeolite.

  7. Combination of Successive Alkalinity Producing System (SAPS) and Aeration for Passive Treatment of Highly Acidic Mine Drainage

    Science.gov (United States)

    Oh, C.; Ji, S.

    2015-12-01

    Passive treatment system has been widely used for remediation of mine drainage since its advantage of low installation and maintenance cost. The system, however, has also a disadvantage in assuring remediation and management efficiency if the drainage is highly acidic mine drainage. To remediate acid mine drainage (AMD) especially showing high acidity, passive treatment system which consists of successive alkalinity producing system (SAPS) and subsequent aeration pond was proposed and its mechanisms and efficiency was evaluated in this research. Target AMD was obtained from Waryong coal mine and showed typical characteristics of AMD having high metal concentration and low pH (acidity > 300 mg/L as CaCO3). Four experimental cases were conducted; untreated, treated with SAPS, treated with aeration, treated with SAPS and aeration to compare role and mechanism of each unit. Between organic matter and limestone layer which constitute SAPS, the former eliminated most of Fe(III) and Al in the AMD so that the latter was kept from being clogged by precipitates. Net acidity of the AMD rapidly decreased by supplement of alkalinity at the limestone layer. A primary function of SAPS, producing alkalinity constantly without clogging, was attained due to addition a portion of limestone particle into the organic matter layer. The discharge from SAPS had low ORP and DO values because of an anaerobic environment formed at the organic matter layer although its alkalinity was increased. This water quality was unfavorable for Fe(II) to be oxidized. Installation of aeration pond after SAPS, therefore, could be effective way of enhancing oxidation rate of Fe(II). Among the experimental cases, the combination of SAPS and aeration pond was only able to remediate the AMD. This concluded that to remediate highly acidic mine drainage with passive treatment system, three critical conditions were required; pre-precipitation of Fe(III) and Al at organic matter layer in SAPS, constant alkalinity

  8. Validation of a high-throughput in vitro alkaline elution/rat hepatocyte assay for DNA damage.

    Science.gov (United States)

    Gealy, Robert; Wright-Bourque, Jennifer L; Kraynak, Andrew R; McKelvey, Troy W; Barnum, John E; Storer, Richard D

    2007-04-20

    In vitro alkaline elution is a sensitive and specific short term assay which measures DNA strand breakage in a mammalian test system (primary rat hepatocytes). This lab has previously demonstrated the performance of the assay with known genotoxic and non-genotoxic compounds. The methodology employed has relatively low sample throughput and is labor-intensive, requiring a great deal of manual processing of samples in a format that is not amenable to automation. Here, we present an automated version of the assay. This high-throughput alkaline elution assay (HT-AE) was made possible through 3 key developments: (1) DNA quantitation using PicoGreen and OliGreen fluorescent DNA binding dyes; (2) design and implementation of a custom automation system; and (3) reducing the assay to a 96-well plate format. The assay can now be run with 5-50mg of test compound. HT-AE was validated in a similar manner as the original assay, including assessment of non-genotoxic and non-carcinogenic compounds and evaluation of cytotoxicity to avoid confounding effects of toxicity-associated DNA degradation. The validation test results from compounds of known genotoxic potential were used to set appropriate criteria to classify alkaline elution results for genotoxicity.

  9. A facile synthesis of highly stable multiblock poly(arylene ether)s based alkaline membranes for fuel cells

    Science.gov (United States)

    Jasti, Amaranadh; Shahi, Vinod K.

    2014-12-01

    Herein, we are disclosing simple route for the preparation of alkaline membranes (AMs) based on aminated multiblock poly(arylene ether)s (AMPEs) synthesized by nucleophilic substitution-poly condensation followed by quaternization and alkalization reactions. In this procedure, four quaternary ammonium groups are successfully introduced without use of carcinogenic reagents such as chloromethylmethyl ether (CMME). Hydrophilic/hydrophobic phase separation is responsible for their high hydroxide conductivity (∼150 mS cm-1 at 80 °C) due to development of interconnected ion transport pathway. AMs are exhibiting good alkaline stability due to the presence of two vicinal quaternary ammonium groups and avoid degradation such as Sommelet-Hauser rearrangement and Hofmann elimination. Vicinal quaternary ammonium groups also resist nucleophilic (OH-) attack and suppress the Stevens rearrangement as well as SN2 substitution reaction due to stearic hindrance. Optimized AM (AMPE-M20N15 (55% DCM)) exhibits about 0.95 V open circuit voltage (OCV) and 48.8 mW cm-2 power density at 65 °C in alkaline direct methanol fuel cell (ADMFC) operation. These results suggest promising begin for the preparation of stable and conductive AMs for ADMFC applications and useful for developing hydroxide conductive materials.

  10. Facile Alkaline Lysis of Escherichia coli Cells in High-Throughput Mode for Screening Enzyme Mutants: Arylsulfatase as an Example.

    Science.gov (United States)

    Yuan, Mei; Yang, Xiaolan; Li, Yuwei; Liu, Hongbo; Pu, Jun; Zhan, Chang-Guo; Liao, Fei

    2016-06-01

    Facile alkaline lysis of Escherichia coli cells in high-throughput (HTP) mode for screening enzyme mutants was tested with Pseudomonas aeruginosa arylsulfatase (PAAS). The alkaline lysis buffer was 1.0 M Tris-HCl at pH 9.0 plus 0.1 % Tween-20 and 2.0 mM 4-aminobenzamidine, mixed with cell suspension at 8:1 to 12:1 ratio for continuous agitation of mixtures in 96-well plates under room temperature; enzymatic activity in lysates was measured with 96-well microplate. PAAS activity tolerated final 0.1 % Tween-20. Individual clones were amplified for 12 h in 0.50 mL TB medium with 48-well plates to enhance the repeatability of induced expression. During continuous agitation of the mixture of cells and the lysis buffer, PAAS activities in lysates were steady from 3 to 9 h and comparable to sonication treatment but better than freezing-thawing. Coefficients of variation of activities of PAAS/mutants in lysates after treatment for 7 h reached ∼22 %. The mutant M72Q had specific activity 2-fold of G138S. By HTP lysis of cells, M72Q was recognized as a positive mutant over G138S with the area under the curve of 0.873. Therefore, for enzymes tolerating concentrated alkaline buffers, the proposed alkaline lysis approach may be generally applicable for HTP lysis of host cells during directed evolution.

  11. OPTIMIZATION OF PROTEASE PRODUCTION FROM FUNGI ISOLATED FROM SOIL

    Directory of Open Access Journals (Sweden)

    Sonia Sethi

    2015-07-01

    Full Text Available Fungal strains isolated from soil by serial dilution method were screened for alkaline protease production. Isolate Penicillium chrysogenum the most potent producer of alkaline protease was identified. The isolate showed highest activity in the optimized medium at pH 9.0, temperature 35ºC, with 1% soycake and peptone incubated for 7 days. Proteases represent one of the largest groups of industrial enzymes and find application in detergents, leather industry, food industry, pharmaceutical industry and bioremediation processes.

  12. High external pH enables more efficient secretion of alkaline α-amylase AmyK38 by Bacillus subtilis

    Directory of Open Access Journals (Sweden)

    Manabe Kenji

    2012-06-01

    Full Text Available Abstract Background Bacillus subtilis genome-reduced strain MGB874 exhibits enhanced production of exogenous extracellular alkaline cellulase Egl-237 and subtilisin-like alkaline protease M-protease. Here, we investigated the suitability of strain MGB874 for the production of α-amylase, which was anticipated to provoke secretion stress responses involving the CssRS (Control secretion stress Regulator and Sensor system. Results Compared to wild-type strain 168, the production of a novel alkaline α-amylase, AmyK38, was severely decreased in strain MGB874 and higher secretion stress responses were also induced. Genetic analyses revealed that these phenomena were attributable to the decreased pH of growth medium as a result of the lowered expression of rocG, encoding glutamate dehydrogenase, whose activity leads to NH3 production. Notably, in both the genome-reduced and wild-type strains, an up-shift of the external pH by the addition of an alkaline solution improved AmyK38 production, which was associated with alleviation of the secretion stress response. These results suggest that the optimal external pH for the secretion of AmyK38 is higher than the typical external pH of growth medium used to culture B. subtilis. Under controlled pH conditions, the highest production level (1.08 g l-1 of AmyK38 was obtained using strain MGB874. Conclusions We demonstrated for the first time that RocG is an important factor for secretory enzyme production in B. subtilis through its role in preventing acidification of the growth medium. As expected, a higher external pH enabled a more efficient secretion of the alkaline α-amylase AmyK38 in B. subtilis. Under controlled pH conditions, the reduced-genome strain MGB874 was demonstrated to be a beneficial host for the production of AmyK38.

  13. Highly Efficient Gluten Degradation by Lactobacilli and Fungal Proteases during Food Processing: New Perspectives for Celiac Disease▿

    Science.gov (United States)

    Rizzello, Carlo G.; De Angelis, Maria; Di Cagno, Raffaella; Camarca, Alessandra; Silano, Marco; Losito, Ilario; De Vincenzi, Massimo; De Bari, Maria D.; Palmisano, Francesco; Maurano, Francesco; Gianfrani, Carmen; Gobbetti, Marco

    2007-01-01

    Presently, the only effective treatment for celiac disease is a life-long gluten-free diet. In this work, we used a new mixture of selected sourdough lactobacilli and fungal proteases to eliminate the toxicity of wheat flour during long-time fermentation. Immunological (R5 antibody-based sandwich and competitive enzyme-linked immunosorbent assay [ELISA] and R5 antibody-based Western blot), two-dimensional electrophoresis, and mass spectrometry (matrix-assisted laser desorption ionization-time of flight, strong-cation-exchange-liquid chromatography/capillary liquid chromatography-electrospray ionization-quadrupole-time of flight [SCX-LC/CapLC-ESI-Q-TOF], and high-pressure liquid chromatography-electrospray ionization-ion trap mass spectrometry) analyses were used to determine the gluten concentration. Assays based on the proliferation of peripheral blood mononuclear cells (PBMCs) and gamma interferon production by PBMCs and intestinal T-cell lines (iTCLs) from 12 celiac disease patients were used to determine the protein toxicity of the pepsin-trypsin digests from fermented wheat dough (sourdough). As determined by R5-based sandwich and competitive ELISAs, the residual concentration of gluten in sourdough was 12 ppm. Albumins, globulins, and gliadins were completely hydrolyzed, while ca. 20% of glutenins persisted. Low-molecular-weight epitopes were not detectable by SCX-LC/CapLC-ESI-Q-TOF mass spectrometry and R5-based Western blot analyses. The kinetics of the hydrolysis of the 33-mer by lactobacilli were highly efficient. All proteins extracted from sourdough activated PBMCs and induced gamma interferon production at levels comparable to the negative control. None of the iTCLs demonstrated immunoreactivity towards pepsin-trypsin digests. Bread making was standardized to show the suitability of the detoxified wheat flour. Food processing by selected sourdough lactobacilli and fungal proteases may be considered an efficient approach to eliminate gluten toxicity

  14. Highly efficient gluten degradation by lactobacilli and fungal proteases during food processing: new perspectives for celiac disease.

    Science.gov (United States)

    Rizzello, Carlo G; De Angelis, Maria; Di Cagno, Raffaella; Camarca, Alessandra; Silano, Marco; Losito, Ilario; De Vincenzi, Massimo; De Bari, Maria D; Palmisano, Francesco; Maurano, Francesco; Gianfrani, Carmen; Gobbetti, Marco

    2007-07-01

    Presently, the only effective treatment for celiac disease is a life-long gluten-free diet. In this work, we used a new mixture of selected sourdough lactobacilli and fungal proteases to eliminate the toxicity of wheat flour during long-time fermentation. Immunological (R5 antibody-based sandwich and competitive enzyme-linked immunosorbent assay [ELISA] and R5 antibody-based Western blot), two-dimensional electrophoresis, and mass spectrometry (matrix-assisted laser desorption ionization-time of flight, strong-cation-exchange-liquid chromatography/capillary liquid chromatography-electrospray ionization-quadrupole-time of flight [SCX-LC/CapLC-ESI-Q-TOF], and high-pressure liquid chromatography-electrospray ionization-ion trap mass spectrometry) analyses were used to determine the gluten concentration. Assays based on the proliferation of peripheral blood mononuclear cells (PBMCs) and gamma interferon production by PBMCs and intestinal T-cell lines (iTCLs) from 12 celiac disease patients were used to determine the protein toxicity of the pepsin-trypsin digests from fermented wheat dough (sourdough). As determined by R5-based sandwich and competitive ELISAs, the residual concentration of gluten in sourdough was 12 ppm. Albumins, globulins, and gliadins were completely hydrolyzed, while ca. 20% of glutenins persisted. Low-molecular-weight epitopes were not detectable by SCX-LC/CapLC-ESI-Q-TOF mass spectrometry and R5-based Western blot analyses. The kinetics of the hydrolysis of the 33-mer by lactobacilli were highly efficient. All proteins extracted from sourdough activated PBMCs and induced gamma interferon production at levels comparable to the negative control. None of the iTCLs demonstrated immunoreactivity towards pepsin-trypsin digests. Bread making was standardized to show the suitability of the detoxified wheat flour. Food processing by selected sourdough lactobacilli and fungal proteases may be considered an efficient approach to eliminate gluten toxicity.

  15. 糯米蛋白提取工艺优化及其水解物性质的测定%Optimization of alkaline protease extraction of sticky rice protein from sticky rice and properties mensuration of hydrolysates of sticky rice protein

    Institute of Scientific and Technical Information of China (English)

    王国泽; 袁怀波; 徐建文

    2012-01-01

    以糯米为原料,采用碱性蛋白酶水解法提取了糯米蛋白。就影响糯米蛋白提取率的5个因素:料液比(g/mL)、加酶量(E/S)、pH、温度和提取时间进行单因素实验和正交实验。研究确立了提取糯米蛋白的最佳工艺条件为料液比1:12,加酶量2%,pH9,温度45℃,提取时间3h。在此条件下,糯米蛋白的提取率可达8.529%。实验表明,糯米蛋白水解物是一类天然的抗氧化氨基酸及其聚合物。%In order to obtain the extraction of sticky rice protein, sticky rice was used as raw materials by alkaline protease .The influence factors, amount of solid to liquid ratio, enzyme dosage, pH, temperature and time of the alkaline protease,which could affect the efficiency of extracting sticky rice protein,were investigated by singlefactor experiment and orthogonal test.The research results showed that the optimal technical parameters could be obtained as following : solid to liquid ratio 1: ]2, enzyme dosage 2%, pH = 9, temperature 45 ℃ and time 3 h.The max extraction rate of sticky rice protein reached to 8.529%.Antioxidant tests of protein hydrolysates by papain showed that hydrolyzate from sticky rice protein serves as amino acids or their polymer.

  16. Compact, Programmable, and Stable Biofunctionalized Upconversion Nanoparticles Prepared through Peptide-Mediated Phase Transfer for High-Sensitive Protease Sensing and in Vivo Apoptosis Imaging.

    Science.gov (United States)

    Zeng, Tao; Zhang, Tao; Wei, Wei; Li, Zhi; Wu, Dan; Wang, Li; Guo, Jun; He, Xuewen; Ma, Nan

    2015-06-10

    Protease represents an important class of biomarkers for disease diagnostics and drug screening. Conventional fluorescence-based probes for in vivo protease imaging suffer from short excitation wavelengths and poor photostability. Upconversion nanoparticles (UCNPs) hold great promise for biosensing and bioimaging because of their deep-tissue excitability, robust photostability, and minimal imaging background. However, producing highly stable and compact biofunctionalized UCNP probes with optimal bioresponsivity for in vivo imaging of protease activities still remains challenging and has not been previously demonstrated. Herein, we report facile preparation of highly compact and stable biofunctionalized UCNPs through peptide-mediated phase transfer for high-sensitive detection of protease in vitro and in vivo. We demonstrate that the polyhistidine-containing chimeric peptides could displace oleic acid molecules capped on UCNPs synthesized in organic solvents and, thereby, directly transfer UCNPs from the chloroform phase to the water phase. The resulting UCNPs possess high stability, programmable surface properties, and a compact coating layer with minimized thickness for efficient luminescence resonance energy transfer (LRET). On the basis of this strategy, we prepared LRET-based UCNP probes with optimal bioresponsivity for in vitro high-sensitive detection of trypsin and in vivo imaging of apoptosis for chemotherapy efficacy evaluation. The reported strategy could be extended to construct a variety of peptide-functionalized UCNPs for various biomedical applications.

  17. Bothrops protease A, a unique highly glycosylated serine proteinase, is a potent, specific fibrinogenolytic agent.

    Science.gov (United States)

    Paes Leme, A F; Prezoto, B C; Yamashiro, E T; Bertholim, L; Tashima, A K; Klitzke, C F; Camargo, A C M; Serrano, S M T

    2008-08-01

    The hemostatic system is the major target of snake venom serine proteinases (SVSPs) that act on substrates of the coagulation, fibrinolytic and kallikrein-kinin systems. Bothrops protease A (BPA), the most glycosylated SVSP, is a non-coagulant, thermostable enzyme. A cDNA encoding BPA showed that the protein has a calculated molecular mass of 25 409 Da, implying that approximately 62% of its molecular mass as assessed by sodium dodecylsulfate polyacrylamide gel electrophoresis (67 kDa) is due to carbohydrate moieties. Here we show that BPA is a potent fibrinogenolytic agent in vitro, as it readily degraded human and rat fibrinogen at a very low enzyme concentration. Partially N-deglycosylated BPA (p-N-d-BPA) generated similar fibrinogen products, but with enhanced fibrinogenolytic activity. In vivo, injection of 0.75 nmoles of BPA in rats completely avoided thrombus formation induced by stasis in the vena cava, or by endothelium injury in the jugular vein. Moreover, it decreased the fibrinogen plasma level and prolonged the recalcification time. Cleavage of fibrinogen in human and rat plasma was observed with native BPA and p-N-d-BPA by electrophoresis followed by western blot using an anti-fibrinogen antibody. BPA did not cause unspecific degradation of plasma proteins and did not cleave isolated albumin, vitronectin and fibronectin at the same concentration used with fibrinogen. Serine proteinase inhibitors failed to inhibit BPA, probably due to steric hindrance caused by its huge carbohydrate moieties. To the best of our knowledge, this investigation underscores a new, thermostable, specific defibrinogenating agent that may have an application in the prevention of thrombus formation.

  18. High power density alkaline fuel cell technology for MMW space burst power

    Science.gov (United States)

    Preston, J. Lawrence, Jr.; Trocciola, John C.; Wertheim, Ronald J.

    The use of advanced alkaline regenerative fuel cell energy storage systems to provide 10's to 100's of MWe of sprint (burst) power for 100's of seconds per orbit of SDI weapons platform was studied. Recharge power is supplied by a multimegawatt space based nuclear power system. Regenerative fuel cell energy storage systems offer the potential for significant platform mass reduction by reducing the size and mass of the nuclear power source required. This is because the reactor can be sized for the smaller average power level for the energy storage system, rather than the sprint power level. The regenerative fuel cell is a particularly attractive energy storage device because the fuel cell is essentially a static power conversion device, which results in excellent platform stability for weapon pointing and tracking. Based upon the detailed point design and conceptual layout, the alkaline regenerative fuel cell energy storage system is an attractive choice for integration with a nuclear thermionic system for providing multimegawatt burst power and multi orbit capability.

  19. Bone mineral density changes in protease inhibitor-sparing vs. nucleoside reverse transcriptase inhibitor-sparing highly active antiretroviral therapy: data from a randomized trial

    DEFF Research Database (Denmark)

    Hansen, Ab; Obel, N; Nielsen, Henrik Ib

    2011-01-01

    Objective The aim of the study was to compare changes in bone mineral density (BMD) over 144 weeks in HIV-infected patients initiating nucleoside reverse transcriptase inhibitor (NRTI)-sparing or protease inhibitor-sparing highly active antiretroviral therapy (HAART). Methods Sixty-three HAART...

  20. Changes in the bacterial populations of the highly alkaline saline soil of the former lake Texcoco (Mexico) following flooding.

    Science.gov (United States)

    Valenzuela-Encinas, César; Neria-González, Isabel; Alcántara-Hernández, Rocio J; Estrada-Alvarado, Isabel; Zavala-Díaz de la Serna, Francisco Javier; Dendooven, Luc; Marsch, Rodolfo

    2009-07-01

    Flooding an extreme alkaline-saline soil decreased alkalinity and salinity, which will change the bacterial populations. Bacterial 16S rDNA libraries were generated of three soils with different electrolytic conductivity (EC), i.e. soil with EC 1.7 dS m(-1) and pH 7.80 (LOW soil), with EC 56 dS m(-1) and pH 10.11 (MEDIUM soil) and with EC 159 dS m(-1) and pH 10.02 (HIGH soil), using universal bacterial oligonucleotide primers, and 463 clone 16S rDNA sequences were analyzed phylogenetically. Library proportions and clone identification of the phyla Proteobacteria, Actinobacteria, Acidobacteria, Cyanobacteria, Bacteroidetes, Firmicutes and Cloroflexi showed that the bacterial communities were different. Species and genera of the Rhizobiales, Rhodobacterales and Xanthomonadales orders of the alpha- and gamma-subdivision of Proteobacteria were found at the three sites. Species and genera of the Rhodospirillales, Sphingobacteriales, Clostridiales, Oscillatoriales and Caldilineales were found only in the HIGH soil, Sphingomonadales, Burkholderiales and Pseudomonadales in the MEDIUM soil, Myxococcales in the LOW soil, and Actinomycetales in the MEDIUM and LOW soils. It was found that the largest diversity at the order and species level was found in the MEDIUM soil as bacteria of both the HIGH and LOW soils were found in it.

  1. Ultra-high performance supercritical fluid chromatography of lignin-derived phenols from alkaline cupric oxide oxidation.

    Science.gov (United States)

    Sun, Mingzhe; Lidén, Gunnar; Sandahl, Margareta; Turner, Charlotta

    2016-08-01

    Traditional chromatographic methods for the analysis of lignin-derived phenolic compounds in environmental samples are generally time consuming. In this work, an ultra-high performance supercritical fluid chromatography method with a diode array detector for the analysis of major lignin-derived phenolic compounds produced by alkaline cupric oxide oxidation was developed. In an analysis of a collection of 11 representative monomeric lignin phenolic compounds, all compounds were clearly separated within 6 min with excellent peak shapes, with a limit of detection of 0.5-2.5 μM, a limit of quantification of 2.5-5.0 μM, and a dynamic range of 5.0-2.0 mM (R(2) > 0.997). The new ultra-high performance supercritical fluid chromatography method was also applied for the qualitative and quantitative analysis of lignin-derived phenolic compounds obtained upon alkaline cupric oxide oxidation of a commercial humic acid. Ten out of the previous eleven model compounds could be quantified in the oxidized humic acid sample. The high separation power and short analysis time obtained demonstrate for the first time that supercritical fluid chromatography is a fast and reliable technique for the analysis of lignin-derived phenols in complex environmental samples.

  2. Highly Dispersed Palladium Nanoparticles on Functional MWNT Surfaces for Methanol Oxidation in Alkaline Solutions

    Institute of Scientific and Technical Information of China (English)

    WANG zhe; ZHU Zan-Zan; LI You-Xiang; LI Hu-Lin

    2008-01-01

    Palladium nanoparticles were crystallized on 4-aminobenzoic acid monolayer-grafted multi-walled carbon nanotubes (MWNT) by diazotization. The structure and nature of the resulting Pd/MWNT composite were characterized by transmission electron microscopy and X-ray diffraction, the results show that the chemically synthesized Pd nanoparticles were homogeneously dispersed and well-separated from one another on the modified MWNT surfaces. Cyclic voltammogram showed that the Pd-MWNT composite materials performed higher electrocatalytic activity and better long-term stability toward methanol oxidation in alkaline solution than Pd-C. The results imply that the Pd-MWNT composite materials as a promising support material improve the excellent electrocatalytic activity for methanol oxidation greatly. So the Pd/MWNT composites have a good application potential to fuel cells.

  3. A binary palladium-bismuth nanocatalyst with high activity and stability for alkaline glucose electrooxidation

    Science.gov (United States)

    Chen, Cheng-Chuan; Lin, Cheng-Lan; Chen, Lin-Chi

    2015-08-01

    Binary palladium-bismuth nanocatalysts supported on functionalized multi-walled carbon nanotubes (Pd-Bi/C) are synthesized using a one-pot polyol method. The prepared Pd-Bi/C catalysts have a metal particle range from 5.25 to 12.98 nm and are investigated for alkaline electrocatalytic glucose oxidation reaction (GOR). The physical properties of the catalysts are characterized by X-ray diffraction (XRD), scanning electron microscopy with energy-dispersive X-ray spectroscopy (SEM-EDS), transmission electron microscopy (TEM) and X-ray photoelectron spectroscopy (XPS). The electrochemical activities are determined by cyclic voltammetry (CV), linear sweep voltammetry (LSV), Tafel analysis and chronoamperomtry (CA) for comparing the electrochemical active surface area (ECSA), GOR onset potential, GOR peak current density, Tafel slope, poisoning rate and cycling stability of the Pd-Bi/C catalysts. It is found that Pd-Bi/C (1:0.14) can significantly enhance the electrocatalytic activity on GOR about 40% times higher than Pd/C and as well as has a 3.7-fold lower poisoning rate. The in-use stability of Pd-Bi/C (1:0.14) is also remarkably improved, according to the results of the 200 cycling CV test. The effects of the operating temperature and the concentration of glucose and NaOH electrolyte on Pd-Bi/C (1:0.14) are further studied in this work. The highest Pd-Bi/C catalyzed GOR current density of 29.5 mA cm-2 is attained in alkaline medium.

  4. Chronic high glucose inhibits albumin reabsorption by lysosomal alkalinization in cultured porcine proximal tubular epithelial cells (LLC-PK1).

    Science.gov (United States)

    Ishibashi, Fukashi

    2006-06-01

    Lysosomal acidification is a key step of albumin reabsorption in proximal tubular epithelial cells (PTECs). This study was performed to examine the influence of chronic high glucose on lysosomal acidification in cultured PTECs. Porcine PTECs (LLC-PK(1) cells) were cultured in 16.7 mM (300 mg/dl) glucose (HG) alone or with 0.5 mM phlorizin for 24 weeks and subsequently for 12 weeks in 5.5 mM (100 mg/dl) glucose (NG). Chronic HG inhibited the fluorescein isothiocyanate (FITC)-albumin (A) uptake progressively, while phlorizin reversed the inhibition. NG for 12 weeks after HG normalized the uptake. The time-dependent uptake of FITC-A was inhibited by HG and bafilomycin A(1) (BafA(1)) after 15 min and by 4,4'-diisothiocyanato-2,2'-disulfonic acid (DIDS) and N-ethyl-N-isopropyl-amiloride (EIPA) after 3 min. Cellular ATP was depleted by HG and restored by NG. Lysosomal pH, assessed by an acidotropic fluorescent probe, was alkalinized (pH 4.5-7.8) with 5.5-27.8 mM glucose and normalized by subsequent NG. BafA(1) alkalinized lysosomes, and the concentration required to 50% change for the pH and 50% inhibition of FITC-A uptake was similar. EIPA inhibited FITC-A uptake, but did not influence lysosomal pH. DIDS inhibited FITC-A uptake, and unexpectedly lowered lysosomal pH. Real time PCR showed that HG reduced the mRNA level for vacuolar H(+)-ATPase, but did not alter those of chloride channel-5 and Na(+)-H(+)-exchanger-3. In conclusion, the chronic HG inhibits albumin reabsorption by lysosomal alkalinization in PTECs, probably due to ATP depletion and down-regulation of vacuolar H(+)-ATPase.

  5. Microbial impacts on (99m)Tc migration through sandstone under highly alkaline conditions relevant to radioactive waste disposal.

    Science.gov (United States)

    Smith, Sarah L; Boothman, Christopher; Williams, Heather A; Ellis, Beverly L; Wragg, Joanna; West, Julia M; Lloyd, Jonathan R

    2017-01-01

    Geological disposal of intermediate level radioactive waste in the UK is planned to involve the use of cementitious materials, facilitating the formation of an alkali-disturbed zone within the host rock. The biogeochemical processes that will occur in this environment, and the extent to which they will impact on radionuclide migration, are currently poorly understood. This study investigates the impact of biogeochemical processes on the mobility of the radionuclide technetium, in column experiments designed to be representative of aspects of the alkali-disturbed zone. Results indicate that microbial processes were capable of inhibiting (99m)Tc migration through columns, and X-ray radiography demonstrated that extensive physical changes had occurred to the material within columns where microbiological activity had been stimulated. The utilisation of organic acids under highly alkaline conditions, generating H2 and CO2, may represent a mechanism by which microbial processes may alter the hydraulic conductivity of a geological environment. Column sediments were dominated by obligately alkaliphilic H2-oxidising bacteria, suggesting that the enrichment of these bacteria may have occurred as a result of H2 generation during organic acid metabolism. The results from these experiments show that microorganisms are able to carry out a number of processes under highly alkaline conditions that could potentially impact on the properties of the host rock surrounding a geological disposal facility for intermediate level radioactive waste. Copyright © 2016. Published by Elsevier B.V.

  6. Optimized coagulation of high alkalinity, low temperature and particle water: pH adjustment and polyelectrolytes as coagulant aids.

    Science.gov (United States)

    Yu, Jianfeng; Wang, Dongsheng; Yan, Mingquan; Ye, Changqing; Yang, Min; Ge, Xiaopeng

    2007-08-01

    The Yellow River in winter as source water is characterized as high alkalinity, low temperature and low particle concentrations, which have brought many difficulties to water treatment plants. This study fully examines the optimized coagulation process of the Yellow River by conventional and pre-polymerized metal coagulants, pH adjustment and polyelectrolytes as the primary coagulants or coagulant aids. For all the metal coagulants, polyaluminum chlorides are superior to traditional metal coagulants due to their stable polymeric species and low consumption of alkalinity. The removal of natural organic matter by monomeric metal coagulants can be improved through pH adjustment, which is in accordance with the higher concentration of polymeric species formed at corresponding pH value. With the addition of polyelectrolytes as coagulant aids, the coagulation performance is significantly improved. The effective removal of dissolved organic matter is consistent with high charge density, while molecular weight is relatively important for removing particles, which is consistent with polyelectrolytes as primary coagulants. These results suggest that the coagulation mechanisms in the removal of dissolved organic matter and particles are different, which may be exploited for optimized coagulation for the typical source water in practice.

  7. Highly Dynamic Interactions Maintain Kinetic Stability of the ClpXP Protease During the ATP-Fueled Mechanical Cycle.

    Science.gov (United States)

    Amor, Alvaro J; Schmitz, Karl R; Sello, Jason K; Baker, Tania A; Sauer, Robert T

    2016-06-17

    The ClpXP protease assembles in a reaction in which an ATP-bound ring hexamer of ClpX binds to one or both heptameric rings of the ClpP peptidase. Contacts between ClpX IGF-loops and clefts on a ClpP ring stabilize the complex. How ClpXP stability is maintained during the ATP-hydrolysis cycle that powers mechanical unfolding and translocation of protein substrates is poorly understood. Here, we use a real-time kinetic assay to monitor the effects of nucleotides on the assembly and disassembly of ClpXP. When ATP is present, complexes containing single-chain ClpX assemble via an intermediate and remain intact until transferred into buffers containing ADP or no nucleotides. ATP binding to high-affinity subunits of the ClpX hexamer prevents rapid dissociation, but additional subunits must be occupied to promote assembly. Small-molecule acyldepsipeptides, which compete with the IGF loops of ClpX for ClpP-cleft binding, cause exceptionally rapid dissociation of otherwise stable ClpXP complexes, suggesting that the IGF-loop interactions with ClpP must be highly dynamic. Our results indicate that the ClpX hexamer spends almost no time in an ATP-free state during the ATPase cycle, allowing highly processive degradation of protein substrates.

  8. Isolation, identification and characterization of organic solvent tolerant protease from Bacillus sp. DAF-01

    Directory of Open Access Journals (Sweden)

    Arastoo Badoei-Dalfard

    2012-01-01

    Full Text Available Introduction: Organic solvent-tolerant bacteria are relatively novel extermophilic microorganisms, which can produce organic tolerant protease with capacity of being used in industrial biotechnology for producing high-value compounds. Therefore, finding of these bacteria has drawn much researchers attention nowadays. Materials and Methods: In this project, samples were collected from a hot spring, located in Jiroft. Samples were incubated in medium supplemented with cyclohexane and toluene for 3 days. Screening of protease producing bacteria was performed on the specific media, SKM (Skim milk agar, based on clear area diameter. The best bacterium was identified based on 16s rDNA gene. Protease activity was considered in different temperatures, pH and organic solvents.Results: Sequence alignment and phylogenetic tree results showed that this bacteria was closely related to Bacillus niacini, with 97% homology. Enzymatic studies showed that, this enzyme was active at a wide range of temperatures, 20-90 °C and it,s optimal activity was in 60 °C. In addition, maximum protease activity was obtained in the 8-9 range of pH, and optimal stability was also at pH 9.0. Protease activity in the presence of methanol, toluene, isopropanol, cyclohexane and DMF ‏showed that, remaining activity was at least 80% compared to the control (without organic solvent Discussion and Conclusion: Thermopilic capacity, being active in alkaline protease and high protease stability in the presence of organic solvents all herald a remarkable application for using in different industries.

  9. Insights from the genome of a high alkaline cellulase producing Aspergillus fumigatus strain obtained from Peruvian Amazon rainforest.

    Science.gov (United States)

    Paul, Sujay; Zhang, Angel; Ludeña, Yvette; Villena, Gretty K; Yu, Fengan; Sherman, David H; Gutiérrez-Correa, Marcel

    2017-06-10

    Here, we report the complete genome sequence of a high alkaline cellulase producing Aspergillus fumigatus strain LMB-35Aa isolated from soil of Peruvian Amazon rainforest. The genome is ∼27.5mb in size, comprises of 228 scaffolds with an average GC content of 50%, and is predicted to contain a total of 8660 protein-coding genes. Of which, 6156 are with known function; it codes for 607 putative CAZymes families potentially involved in carbohydrate metabolism. Several important cellulose degrading genes, such as endoglucanase A, endoglucanase B, endoglucanase D and beta-glucosidase, are also identified. The genome of A. fumigatus strain LMB-35Aa represents the first whole sequenced genome of non-clinical, high cellulase producing A. fumigatus strain isolated from forest soil. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. Mutations affecting extracellular protease production in the filamentous fungus Aspergillus nidulans.

    Science.gov (United States)

    Katz, M E; Flynn, P K; vanKuyk, P A; Cheetham, B F

    1996-04-10

    The extracellular proteases of Aspergillus nidulans are known to be regulated by carbon, nitrogen and sulphur metabolite repression. In this study, a mutant with reduced levels of extracellular protease was isolated by screening for loss of halo production on milk plates. Genetic analysis of the mutant showed that it contains a single, recessive mutation, in a gene which we have designated xprE, located on chromosome VI. The xprE1 mutation affected the production of extracellular proteases in response to carbon, nitrogen and, to a lesser extent, sulphur limitation. Three reversion mutations, xprF1, xprF2 and xprG1, which suppress xprE1, were characterised. Both xprF and xprG map to chromosome VII but the two genes are unlinked. The xprF1, xprF2 and xprG1 mutants showed high levels of milk-clearing activity on medium containing milk as a carbon source but reduced growth on a number of nitrogen sources. Evidence is presented that the xprE1 and xprG1 mutations alter expression of more than one protease and affect levels of alkaline protease gene mRNA.

  11. Hydrolysis of Fish Protein by Analkaline Protease

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Cod muscle protein was hydrolyzed by an alkaline protease in our study. The influences of hydrolysis temperature,fish protein concentration,and ratio of protease addition to protein amount on its degree of hy drolysis (DH) of protein were studied in details by applying dual quadratic rotary combinational design. The final results showed that more than 84% cod muscle protein could be hydrolyzed and recovered. Cod protein hydrolysate thus obtained had a balanced amino acid composition and mainly consisted of small peptides with molecule weight less than 6900 dalton.

  12. Proteases as Insecticidal Agents

    OpenAIRE

    Robert L. Harrison; Bonning, Bryony C.

    2010-01-01

    Proteases from a variety of sources (viruses, bacteria, fungi, plants, and insects) have toxicity towards insects. Some of these insecticidal proteases evolved as venom components, herbivore resistance factors, or microbial pathogenicity factors, while other proteases play roles in insect development or digestion, but exert an insecticidal effect when over-expressed from genetically engineered plants or microbial pathogens. Many of these proteases are cysteine proteases, although insect-toxic...

  13. Study of the catalytic properties of bacillus subtilis proteases Estudio de las propiedades catalíticas de las proteasas bacillus subtilis

    Directory of Open Access Journals (Sweden)

    Salcedo L.

    1998-06-01

    Full Text Available The catalytic properties of proteases isolated from the filtrate of submerged fermentation of Bacillus subtilis were investigated. Proteases present in the filtrate were determined to be of the serine protease type based on the use of specific protease inhibitors; ethylenediamintetraacetic acid (EDTA was used as a metalloprotease inhibitor, and phenylmethylsulfonylfluoride (PMSF was used as a serine protease inhibitor. Protease activity was highly stable in alkaline solutions and at high temperatures as well as in the presence of detergents. We propose that this protease preparation be used as biocomponent in detergent production.Se investigaron las propiedades catalíticas de las proteasas obtenidas del filtrado de cultivo de la bacteria Bacillus subtilis. Utilizando inhibidores específicos de proteasas se determinó que las proteasas presentes en el filtrado pertenecían al grupo de las serina proteasas. Se utilizó ácido etilendiaminatetraacético (EDTA como inhibidor de metaloproteasas, y fenilmetilsulfonil fluoruro (FMSF como inhibidor de serina proteasas. La actividad proteolítica fue altamente estable en soluciones alcalinas y a altas temperaturas, además tolero la presencia de detergentes. Se propone que estas proteasas sean utilizadas en calidad de biocomponente para la producción de detergentes.

  14. High-T sub c thin films on low microwave loss alkaline-rare-earth-aluminate crystals

    Energy Technology Data Exchange (ETDEWEB)

    Sobolewski, R.; Gierlowski, P.; Kula, W.; Zarembinski, S.; Lewandowski, S.J.; Berkowski, M.; Pajaczkowska, A. (Instytut Fizyki, Polska Akatlemia Nauk, Al. Lotnikow 32/46, PL-02668 Warszawa (PL)); Gorshunov, B.P.; Lyudmirsky, D.B.; Sirotinsky, O.I. (Institute of General Physics, USSR Academy of Sciences, 38 Vavilova Street, SU-117924 Moscow (SU))

    1991-03-01

    This paper reports on the alkaline-rare-earth aluminates (K{sub 2}NiF{sub 4}-type perovskites) which are an excellent choice as the substrate material for the growth of high-T{sub c} thin films suitable for microwave and far-infrared applications. The CaNdAlO{sub 4}, and SrLaAlO{sub 4} single crystals have been grown by Czochralski pulling and fabricated into the form of (001) oriented wafers. The Y-Ba-Cu-O and Bi-Sr-Ca-Cu-O films deposited on these substrates by a single-target magnetron sputtering exhibited very good superconducting and structural properties.

  15. In-situ potentiostatic activation to optimize electrodeposited cobalt-phosphide electrocatalyst for highly efficient hydrogen evolution in alkaline media

    Science.gov (United States)

    Wei, Mengmeng; Yang, Liming; Wang, Longlu; Liu, Tian; Liu, Chengbin; Tang, Yanhong; Luo, Shenglian

    2017-08-01

    We first report a novel cobalt-phosphide (Co-P) hybrid with flake-like structure by a facile one-step electrodeposition combined with in-situ potentiostatic activation technique. Exotic microstructure transformation of Co-P hybrid from microspheres to nanosheets has been noted during the activation process. The Co-P catalyst exhibits striking kinetic metrics with an overpotential of 85 mV (at 10 mA cm-2) and Tafel slope of 37 mV dec-1, performing among the best of all the HER catalysts in strong alkaline media (at pH 14). This study offers a new in-situ approach to optimize catalytic materials for high-performance electrocatalysts towards energy-related applications.

  16. Diversity and food web structure of nematode communities under high soil salinity and alkaline pH.

    Science.gov (United States)

    Salamún, Peter; Kucanová, Eva; Brázová, Tímea; Miklisová, Dana; Renčo, Marek; Hanzelová, Vladimíra

    2014-10-01

    A long-term and intensive magnesium (Mg) ore processing in Slovenské Magnezitové Závody a.s. in Jelšava has resulted in a high Mg content and alkaline pH of the soil environment, noticeable mainly in the close vicinity of the smelter. Nematode communities strongly reacted to the contamination mostly by a decrease in abundance of the sensitive groups. Nematodes from c-p 1 group and bacterivores, tolerant to pollution played a significant role in establishing the dominance at all sites. With increasing distance from the pollution source, the nematode communities were more structured and complex, with an increase in proportion of sensitive c-p 4 and 5 nematodes, composed mainly of carnivores and omnivores. Various ecological indices (e.g. MI2-5, SI, H') indicated similar improvement of farther soil ecosystems.

  17. The New High Resolution Crystal Structure of NS2B-NS3 Protease of Zika Virus

    Science.gov (United States)

    Badshah, Syed Lal; Naeem, Abdul; Mabkhot, Yahia

    2017-01-01

    Zika virus (ZIKV) is the cause of a significant viral disease affecting humans, which has spread throughout many South American countries and has also become a threat to Southeastern Asia. This commentary discusses the article “Crystal structure of unlinked NS2B-NS3 protease from Zika virus” published recently in the journal Science by Zhang et al. of Nanyang Technological University, Singapore. They resolved a 1.58 Å resolution structure of the NS2B-NS3 protease of ZIKV and demonstrated how peptide and non-peptide inhibitors interact with this structure, along with the different conformational states that were observed. This protease crystal structure offers new opportunities for the design and development of novel antiviral drugs used for the treatment and control of ZIKV. PMID:28075376

  18. The New High Resolution Crystal Structure of NS2B-NS3 Protease of Zika Virus

    OpenAIRE

    Syed Lal Badshah; Abdul Naeem; Yahia Mabkhot

    2017-01-01

    Zika virus (ZIKV) is the cause of a significant viral disease affecting humans, which has spread throughout many South American countries and has also become a threat to Southeastern Asia. This commentary discusses the article “Crystal structure of unlinked NS2B-NS3 protease from Zika virus” published recently in the journal Science by Zhang et al. of Nanyang Technological University, Singapore. They resolved a 1.58 Å resolution structure of the NS2B-NS3 protease of ZIKV and demonstrated how ...

  19. Proteolytic activity of protease produced by Pseudomonas fluorescens IB 2312 in skimmed milk subject to the process of high pressure homogenization

    Directory of Open Access Journals (Sweden)

    Claudia Regina Gonçalves Pinho

    2014-08-01

    Full Text Available The presence of thermoresistant proteases produced by psychrotrophic microorganisms have been identified as a limiting factor of the UHT milk shelflife, causing undesirable changes in milk products. High pressure homogenization (HPH processing is a non-thermal method of food preservation, able to promotes the microbiological safety and inactivation of some enzymes. Thus, this work assessed the proteolytic activity of protease produced by Pseudomonas fluorescens in skim milk subjected to high pressure homogenization process. The milk samples were added by the protease enzymatic extract (10% v/v and subjected to pressures up to 300 MPa. The assays showed that pressures on the order of 300 MPa caused a 72.5% reduction in proteolytic activity. Therefore, the process at high pressures resulted in significant inactivation of this thermoresistent enzyme, which possibly favors the shelf-life extension of the UHT milk and also limits the yield and quality loss of cheeses due to undesirable sensory changes in flavor and texture caused by this enzyme.

  20. Cystatins - Extra- and intracellular cysteine protease inhibitors: High-level secretion and uptake of cystatin C in human neuroblastoma cells

    DEFF Research Database (Denmark)

    Wallin, Hanna; Bjarnadottir, Maria; Vogel, Lotte

    2010-01-01

    Cystatins are present in mammals, birds, fish, insects, plants, fungi and protozoa and constitute a large protein family, with most members sharing a cysteine protease inhibitory function. In humans 12 functional cystatins exist, forming three groups based on molecular organisation and distribution...

  1. High electrocatalytic performance of nitrogen-doped carbon nanofiber-supported nickel oxide nanocomposite for methanol oxidation in alkaline medium

    Science.gov (United States)

    Al-Enizi, Abdullah M.; Elzatahry, Ahmed A.; Abdullah, Aboubakr M.; Vinu, Ajayan; Iwai, Hideo; Al-Deyab, Salem S.

    2017-04-01

    Nitrogen-Doped Carbon Nanofiber (N-CNF)-supported NiO composite was prepared by electrospinning a sol-gel mixture of graphene and polyaniline (PANi) with aqueous solutions of Polyvinylpyrrolidone (PVP) followed by a high-temperature annealing process. The electrospun was stabilized for 2 h at 280 °C, carbonized for 5 h at 1200 °C then loaded by 10% NiO. The electrocatalytic activities of the produced nanocomposite have been studied using cyclic voltammetry, and chronoamperometry. Also, N-CNF was characterized by X-ray diffraction (XRD), thermogravimetric analysis (TGA), surface area (BET), X-ray photoelectron spectroscopy (XPS), transmission electron microscope (TEM), and scanning-electron microscopy (SEM). The obtained N-doped carbon nanofiber was found to have a nitrogen content of 2.6 atomic% with a diameter range of (140-160) nm, and a surface area (393.3 m2 g-1). In addition, it showed a high electrocatalytic behavior towards methanol oxidation reaction in alkaline medium and high stability and resistivity to the adsorption of intermediates.

  2. A High Level of Intestinal Alkaline Phosphatase Is Protective Against Type 2 Diabetes Mellitus Irrespective of Obesity.

    Science.gov (United States)

    Malo, Madhu S

    2015-12-01

    Mice deficient in intestinal alkaline phosphatase (IAP) develop type 2 diabetes mellitus (T2DM). We hypothesized that a high level of IAP might be protective against T2DM in humans. We determined IAP levels in the stools of 202 diabetic patients and 445 healthy non-diabetic control people. We found that compared to controls, T2DM patients have approx. 50% less IAP (mean +/- SEM: 67.4 +/- 3.2 vs 35.3 +/- 2.5 U/g stool, respectively; p diabetes status. With each 25 U/g decrease in stool IAP, there is a 35% increased risk of diabetes. The study revealed that obese people with high IAP (approx. 65 U/g stool) do not develop T2DM. Approx. 65% of the healthy population have diabetes', and might develop T2DM and other metabolic disorders in the near future. In conclusion, high IAP levels appear to be protective against diabetes irrespective of obesity, and a 'temporal IAP profile' might be a valuable tool for predicting 'the incipient metabolic syndrome', including 'incipient diabetes'.

  3. Alkaline Exchange Membrane (AEM) for High-Efficiency Fuel Cells, Electrolyzers and Regenerative Fuel Cell Systems Project

    Data.gov (United States)

    National Aeronautics and Space Administration — Develop an alkaline exchange membrane (AEM)for use as a polymer electrolyte in both fuel cell and electrolyzer systems.  The ultimate goal in AEM development is...

  4. Characterization of proteases from Planomicrobium sp. L-2 isolated from the gastrointestinal tract of Octopus variabilis (Sasaki)

    Science.gov (United States)

    Jin, Yulan; Wang, Yurong; Xiao, Lin; Lin, Xiukun

    2016-05-01

    A crude protease produced from Planomicrobium sp. L-2 is described, and its effectiveness as an additive in liquid detergent evaluated. We isolate the protease-producing Planomicrobium sp. L-2 from the gastrointestinal tract of Octopus variabilis. At least three caseinolytic protease clear bands were observed in zymogram analysis. The crude alkaline protease was highly tolerant of a pH range from 7.0 to 9.0, and temperatures to 50°C after incubation for 1 h. Proteolytic enzymes were stable towards three surfactants (5% Tween 80, 1% Triton X-100 and 0.05% SDS) and an oxidizing agent (1% hydrogen peroxide), in addition to being highly stable and compatible with popular commercial laundry powered detergent brands available in China. Our study demonstrates the potential these proteases have for development into novel classes of detergent additive. This study also suggests that the gastrointestinal tract of Octopus variabilis may be a rich source of commercially valuable strains of enzyme.

  5. High Temperature and Pressure Alkaline Electrochemical Reactor for Conversion of Power to Chemicals

    DEFF Research Database (Denmark)

    Chatzichristodoulou, Christodoulos

    2016-01-01

    Moving away from fossil fuels requires harvesting more and more intermittent renewable energy resources and establishing a sustainable system for the production of chemicals. This brings forward the need for efficient large scale energy storage technologies 1-3 and technologies for the conversion...... of renewable electricity to chemicals. Electrochemical reactors can play a crucial role in this endeavor, since they can efficiently and reversibly transform electricity to high-value chemicals, and thus serve as energy storage and recovery devices for balancing the grid, while offering a means...

  6. Bowman-Birk protease inhibitor from the seeds of Vigna unguiculata forms a highly stable dimeric structure.

    Science.gov (United States)

    Rao, K N; Suresh, C G

    2007-10-01

    Different protease inhibitors including Bowman-Birk type (BBI) have been reported from the seeds of Vigna unguiculata. Protease isoinhibitors of double-headed Bowman-Birk type from the seeds of Vigna unguiculata have been purified and characterized. The BBI from Vigna unguiculata (Vu-BBI) has been found to undergo self-association to form very stable dimers and more complex oligomers, by size-exclusion chromatography and SDS-PAGE in the presence of urea. Many BBIs have been reported to undergo self-association to form homodimers or more complex oligomers in solution. Only one dimeric crystal structure of a BBI (pea-BBI) is reported to date. We report the three-dimensional structure of a Vu-BBI determined at 2.5 A resolution. Although, the inhibitor has a monomer fold similar to that found in other known structures of Bowman-Birk protease inhibitors, its quaternary structure is different from that commonly observed in this family. The structural elements responsible for the stability of monomer molecule and dimeric association are discussed. The Vu-BBI may use dimeric or higher quaternary association to maintain the physiological state and to execute its biological function.

  7. A multi-scale model of the oxygen reduction reaction on highly active graphene nanosheets in alkaline conditions

    Science.gov (United States)

    Vazquez-Arenas, Jorge; Ramos-Sanchez, Guadalupe; Franco, Alejandro A.

    2016-10-01

    A multi-scale model based on a mean field approach, is proposed to describe the ORR mechanism on N-GN catalysts in alkaline media. The model implements activation energies calculated with Density Functional Theory (DFT) at the atomistic level, and scales up them into a continuum framework describing the cathode/electrolyte interface at the mesoscale level. The model also considers mass and momentum transports arising in the region next to the rotating electrode for all ionic species and O2; correction of potential drop and electrochemical double-layer capacitance. Most fitted parameters describing the kinetics of ORR elementary reactions are sensitive in the multi-scale model, which results from the incorporation of activation energies using the mean field method, unlike single-scale modelling Errors in the deviations from activation energies are found to be moderate, except for the elementary step (2) related to the formation of O2ads, which can be assigned to the inherent DFT limitations. The consumption of O2ads to form OOHads is determined as the rate-determining step as a result of its highest energy barrier (163.10 kJ mol-1) in the system, the largest error obtained for the deviation from activation energy (28.15%), and high sensitivity. This finding is confirmed with the calculated surface concentration and coverage of electroactive species.

  8. Tissue non-specific alkaline phosphatase production by human dental pulp stromal cells is enhanced by high density cell culture.

    Science.gov (United States)

    Tomlinson, Matthew J; Dennis, Caitriona; Yang, Xuebin B; Kirkham, Jennifer

    2015-08-01

    The cell surface hydrolase tissue non-specific alkaline phosphatase (TNAP) (also known as MSCA-1) is used to identify a sub-population of bone marrow stromal cells (BMSCs) with high mineralising potential and is found on subsets of cells within the dental pulp. We aim to determine whether TNAP is co-expressed by human dental pulp stromal cells (hDPSCs) alongside a range of BMSC markers, whether this is an active form of the enzyme and the effects of culture duration and cell density on its expression. Cells from primary dental pulp and culture expanded hDPSCs expressed TNAP. Subsequent analyses revealed persistent TNAP expression and co-expression with BMSC markers such as CD73 and CD90. Flow cytometry and biochemical assays showed that increased culture durations and cell densities enhanced TNAP expression by hDPSCs. Arresting the hDPSC cell cycle also increased TNAP expression. These data confirm that TNAP is co-expressed by hDPSCs together with other BMSC markers and show that cell density affects TNAP expression levels. We conclude that TNAP is a potentially useful marker for hDPSC selection especially for uses in mineralised tissue regenerative therapies.

  9. Microprofiles of oxygen, redox potential, and pH, and microbial fermentation products in the highly alkaline gut of the saprophagous larva of Penthetria holosericea (Diptera: Bibionidae).

    Science.gov (United States)

    Šustr, Vladimír; Stingl, Ulrich; Brune, Andreas

    2014-08-01

    The saprophagous larvae of bibionid flies harbor bacteria in their alkaline intestinal tracts, but little is known about the contribution of the gut microbiota to the digestion of their recalcitrant diet. In this study, we measured oxygen and hydrogen partial pressure, redox potential and pH in the midgut, gastric caeca and hindgut of larvae of the bibionid fly Penthetria holosericea with Clark-type O2 and H2 microsensors, platinum redox microelectrodes, and LIX-type pH microelectrodes. The center of the midgut lumen was anoxic, whereas gastric caeca and hindgut were hypoxic. However, redox potential profiles indicated oxidizing conditions throughout the gut, with lowest values in the midgut (+20 to +60mV). Hydrogen production was not detected. The midgut was extremely alkaline (pH around 11), whereas hindgut and gastric caeca were neutral to slightly alkaline. While HPLC analysis showed high concentrations of glucose in the midgut (15mM) and gastric caeca (27mM), the concentrations of microbial fermentation products such as lactate (2-4mM), acetate (larva of P. holosericea, which feeds selectively on decomposed leaves and its own microbe-rich faeces, differs fundamentally from those of detritivorous and humivorous insects, which host a highly active, fermentative microbiota in their alkaline midgut or hindgut compartments.

  10. Technetium in alkaline, high-salt, radioactive tank waste supernate: Preliminary characterization and removal

    Energy Technology Data Exchange (ETDEWEB)

    Blanchard, D.L. Jr.; Brown, G.N.; Conradson, S.D. [and others

    1997-01-01

    This report describes the initial work conducted at Pacific Northwest National Laboratory to study technetium (Tc) removal from Hanford tank waste supernates and Tc oxidation state in the supernates. Filtered supernate samples from four tanks were studied: a composite double shell slurry feed (DSSF) consisting of 70% from Tank AW-101, 20% from AP-106, and 10% from AP-102; and three complexant concentrate (CC) wastes (Tanks AN-107, SY-101, ANS SY-103) that are distinguished by having a high concentration of organic complexants. The work included batch contacts of these waste samples with Reillex{trademark}-HPQ (anion exchanger from Reilly Industries) and ABEC 5000 (a sorbent from Eichrom Industries), materials designed to effectively remove Tc as pertechnetate from tank wastes. A short study of Tc analysis methods was completed. A preliminary identification of the oxidation state of non-pertechnetate species in the supernates was made by analyzing the technetium x-ray absorption spectra of four CC waste samples. Molybdenum (Mo) and rhenium (Re) spiked test solutions and simulants were tested with electrospray ionization-mass spectrometry to evaluate the feasibility of the technique for identifying Tc species in waste samples.

  11. The vacuolar serine protease, a cross-reactive allergen from Cladosporium herbarum.

    Science.gov (United States)

    Pöll, Verena; Denk, Ursula; Shen, Horng-Der; Panzani, Raphael C; Dissertori, Oliver; Lackner, Peter; Hemmer, Wolfgang; Mari, Adriano; Crameri, Reto; Lottspeich, Friedrich; Rid, Raphaela; Richter, Klaus; Breitenbach, Michael; Simon-Nobbe, Birgit

    2009-04-01

    Subtilisin-like serine proteases make up one of the most important allergen-families regarding the number of individual allergens. Previously, fungal subtilisin-like serine proteases have been identified from Aspergillus-, Penicillium-, and Trichophyton-species having a prevalence of IgE-reactivity between 33% and 80%. Since IgE-cross-reactivity is a common phenomenon within fungal species we wanted to know whether this protein also represents an allergen in Cladosporium herbarum. Hence, a screening of a C. herbarum cDNA library was performed using the coding sequence of the Penicillium oxalicum vacuolar serine protease (Pen o 18) as hybridization probe, ending up with a full-length clone. Biochemical and immunological characterization of this clone revealed that C. herbarum vacuolar serine protease most likely is synthesized as a precursor with an N-terminal pro-enzyme sequence and represents a minor allergen (Cla h 9) with a prevalence of IgE-reactivity of 15.5%. Furthermore Cla h 9 specifically reacted with the two monoclonal antibodies FUM20 and PCM39, as do the vacuolar serine proteases from Aspergillus fumigatus and Penicillium species. Investigation of IgE-cross-reactivity between Cla h 9 and other fungal serine proteases revealed that cross-reactivity is higher between vacuolar than alkaline serine proteases. IgE-epitope mapping of Cla h 9 was done in order to test whether four Cla h 9-peptides having a high sequence homology to previously determined Pen ch 18-IgE-epitopes also harbour IgE-epitopes. Three-dimensional models of the vacuolar serine proteases from C. herbarum and Penicillium chrysogenum were generated for the three-dimensional localization of the Cla h 9- and Pen ch 18- IgE-reactive and -non-reactive peptides. Taken together a new C. herbarum allergen has been identified, which may be useful in a molecule-based approach of C. herbarum allergy-diagnosis and -therapy. Moreover, Cla h 9 represents a further member of the subtilisin-like serine

  12. Isolation and characterization of a marine bacterium producing protease from Chukchi Sea, Arctic

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    A Gram negative bacterium Ar/W/b/75°25'N/1 producing extracellular alkaline protease was isolated from surface water of latitude 75°25'N, and longitude 162°25'W in Chukchi sea, Arctic. The strain can grow at the temperature range from 7℃ to 30℃, and grow better at 30(℃. It can not grow at 40℃. Keeping certain salinity concentration in medium is necessary for cell growth. It grows well in medium containing salinity concentration from 0. 5 % to 10 % sodium chloride. Glucose, sucrose and soluble starch can be utilized by the strain, among which glucose is the optimal carbon source. Peptone is the optimal organic nitrogen source for cell growth and protease producing, and ammonium nitrate is the optimal inorganic nitrogen source.About 75.7% of total protease of the strain are extracellular enzyme. Optimal temperature for proteolytic activity is at 40℃. Protease of the strain keeps stable below 40℃, and shows high proteolytic activity within the pH range from 7 to 11.

  13. Cleavage entropy as quantitative measure of protease specificity.

    Science.gov (United States)

    Fuchs, Julian E; von Grafenstein, Susanne; Huber, Roland G; Margreiter, Michael A; Spitzer, Gudrun M; Wallnoefer, Hannes G; Liedl, Klaus R

    2013-04-01

    A purely information theory-guided approach to quantitatively characterize protease specificity is established. We calculate an entropy value for each protease subpocket based on sequences of cleaved substrates extracted from the MEROPS database. We compare our results with known subpocket specificity profiles for individual proteases and protease groups (e.g. serine proteases, metallo proteases) and reflect them quantitatively. Summation of subpocket-wise cleavage entropy contributions yields a measure for overall protease substrate specificity. This total cleavage entropy allows ranking of different proteases with respect to their specificity, separating unspecific digestive enzymes showing high total cleavage entropy from specific proteases involved in signaling cascades. The development of a quantitative cleavage entropy score allows an unbiased comparison of subpocket-wise and overall protease specificity. Thus, it enables assessment of relative importance of physicochemical and structural descriptors in protease recognition. We present an exemplary application of cleavage entropy in tracing substrate specificity in protease evolution. This highlights the wide range of substrate promiscuity within homologue proteases and hence the heavy impact of a limited number of mutations on individual substrate specificity.

  14. Cleavage entropy as quantitative measure of protease specificity.

    Directory of Open Access Journals (Sweden)

    Julian E Fuchs

    2013-04-01

    Full Text Available A purely information theory-guided approach to quantitatively characterize protease specificity is established. We calculate an entropy value for each protease subpocket based on sequences of cleaved substrates extracted from the MEROPS database. We compare our results with known subpocket specificity profiles for individual proteases and protease groups (e.g. serine proteases, metallo proteases and reflect them quantitatively. Summation of subpocket-wise cleavage entropy contributions yields a measure for overall protease substrate specificity. This total cleavage entropy allows ranking of different proteases with respect to their specificity, separating unspecific digestive enzymes showing high total cleavage entropy from specific proteases involved in signaling cascades. The development of a quantitative cleavage entropy score allows an unbiased comparison of subpocket-wise and overall protease specificity. Thus, it enables assessment of relative importance of physicochemical and structural descriptors in protease recognition. We present an exemplary application of cleavage entropy in tracing substrate specificity in protease evolution. This highlights the wide range of substrate promiscuity within homologue proteases and hence the heavy impact of a limited number of mutations on individual substrate specificity.

  15. Short-term effect of vermicompost application on biological properties of an alkaline soil with high lime content from Mediterranean region of Turkey.

    Science.gov (United States)

    Uz, Ilker; Tavali, Ismail Emrah

    2014-01-01

    This study was conducted to investigate direct short-term impact of vermicompost on some soil biological properties by monitoring changes after addition of vermicompost as compared to farmyard manure in an alkaline soil with high lime content from semiarid Mediterranean region of Turkey. For this purpose, mixtures of soil and organic fertilizers in different doses were incubated under greenhouse condition. Soil samples collected in regular intervals were analyzed for biological parameters including dehydrogenase, β-glucosidase, urease, alkaline phosphatase activities, and total number of aerobic mesophilic bacteria. Even though soil dehydrogenase activity appeared to be dose-independent based on overall evaluation, organic amendments were found to elevate dehydrogenase activity when sampling periods are evaluated individually. β-glucosidase, urease, alkaline phosphatase activity, and aerobic mesophilic bacterial numbers in vermicompost treatments fluctuated but remained significantly above the control. A slight but statistically significant difference was detected between organic amendments in terms of urease activity. Vermicompost appeared to more significantly increase bacterial number in soil. Clearly, vermicompost has a potential to be used as an alternative to farmyard manure to improve and maintain soil biological activity in alkaline calcareous soils from the Mediterranean region of Turkey. Further studies are needed to assess its full potential for these soils.

  16. Microprofiles of oxygen, redox potential, and pH, and microbial fermentation products in the highly alkaline gut of the saprophagous larva of Penthetria holosericea (Diptera: Bibionidae)

    KAUST Repository

    Šustr, Vladimír

    2014-08-01

    The saprophagous larvae of bibionid flies harbor bacteria in their alkaline intestinal tracts, but little is known about the contribution of the gut microbiota to the digestion of their recalcitrant diet. In this study, we measured oxygen and hydrogen partial pressure, redox potential and pH in the midgut, gastric caeca and hindgut of larvae of the bibionid fly Penthetria holosericea with Clark-type O2 and H2 microsensors, platinum redox microelectrodes, and LIX-type pH microelectrodes. The center of the midgut lumen was anoxic, whereas gastric caeca and hindgut were hypoxic. However, redox potential profiles indicated oxidizing conditions throughout the gut, with lowest values in the midgut (+20 to +60mV). Hydrogen production was not detected. The midgut was extremely alkaline (pH around 11), whereas hindgut and gastric caeca were neutral to slightly alkaline. While HPLC analysis showed high concentrations of glucose in the midgut (15mM) and gastric caeca (27mM), the concentrations of microbial fermentation products such as lactate (2-4mM), acetate (<1mM) and succinate (<0.5mM) were low in all gut regions, suggesting that the contribution of microorganisms to the digestive process, particularly in the alkaline midgut, is only of minor importance. We conclude that the digestive strategy of the saprophytic larva of P. holosericea, which feeds selectively on decomposed leaves and its own microbe-rich faeces, differs fundamentally from those of detritivorous and humivorous insects, which host a highly active, fermentative microbiota in their alkaline midgut or hindgut compartments. © 2014 Elsevier Ltd.

  17. FeS anchored reduced graphene oxide nanosheets as advanced anode material with superior high-rate performance for alkaline secondary batteries

    Science.gov (United States)

    Shangguan, Enbo; Guo, Litan; Li, Fei; Wang, Qin; Li, Jing; Li, Quanmin; Chang, Zhaorong; Yuan, Xiao-Zi

    2016-09-01

    A new nanocomposite formulation of the iron-based anode for alkaline secondary batteries is proposed. For the first time, FeS nanoparticles anchored on reduced graphene oxide (RGO) nanosheets are synthesized via a facile, environmentally friendly direct-precipitation approach. In this nanocomposite, FeS nanoparticles are anchored uniformly and tightly on the surface of RGO nanosheets. As an alkaline battery anode, the FeS@RGO electrode delivers a superior high-rate charge/discharge capability and outstanding cycling stability, even at a condition without any conductive additives and a high electrode loading of ∼40 mg cm-2. At high charge/discharge rates of 5C, 10C and 20C (6000 mA g-1), the FeS@RGO electrode presents a specific capacity of ∼288, 258 and 220 mAh g-1, respectively. Moreover, the FeS@RGO electrode exhibits an admirable long cycling stability with a superior capacity retention of 87.6% for 300 cycles at a charge/discharge rate of 2C. The excellent electrochemical properties of the FeS@RGO electrode can be stemmed from the high specific surface area, peculiar electric conductivity and robust sheet-anchored structure of the FeS@RGO nanocomposite. By virtue of its superior fast charge/discharge properties, the FeS@RGO nanocomposite is suitable as an advanced anode material for high-performance alkaline secondary batteries.

  18. Control of exocellular proteases in dermatophytes and especially Trichophyton rubrum.

    Science.gov (United States)

    Meevootisom, V; Niederpruem, D J

    1979-06-01

    The production of proteases was investigated during growth of dermatophytic fungi with special emphasis on Trichophyton rubrum. Exogenous glucose suppressed elastase production in all dermatophytes examined. The production of protease active guinea pig hair in keratin-salts broth by Microsporum gypseum. Trichophyton mentagrophytes and T. rubrum was also suppressed by glucose. Various carbohydrates added to keratin-salts broth curtailed protease production by T. rubrum as did individual amino acids but ammonium phosphate did not. Enzyme activities against guinea pig hair were compared in twenty-one diverse clinical isolates of T. rubrum cultured in keratin-salts broth. Activity also occurred towards casein, bovine serum albumin, keratin, collagen and elastin after keratin-growth. Studies concerning the properties of enzyme activities in culture filtrates of T. rubrum after keratin-growth suggested that multiple proteases occurred here. Hydrolysis of guinea pig hair and elastin were optimal at pH7 while keratinase was most active at alkaline pH. Divalent cations stimulated protease(s). Ferric ion and mercuric ion stimulated keratinase but were inhibitory to guinea pig hair hydrolysis and elastase. Chelating agents inhibited elastase and the hydrolysis of guinea pig hair more severely than keratinase and all of those effects were reversed by excess calcium. A serine-protease inhibitor, phenylmethylsulfonylfluoride (PMSF), curtailed keratinase but was less inhibitory to elastase and guinea pig hair hydrolysis. Soybean trypsin inhibitor arrested each protease.

  19. Evaluation of gardenia yellow using crocetin from alkaline hydrolysis based on ultra high performance liquid chromatography and high-speed countercurrent chromatography.

    Science.gov (United States)

    Inoue, Koichi; Tanada, Chihiro; Nishikawa, Hiroaki; Matsuda, Satoru; Tada, Atsuko; Ito, Yusai; Min, Jun Zhe; Todoroki, Kenichiro; Sugimoto, Naoki; Toyo'oka, Toshimasa; Akiyama, Hiroshi

    2014-12-01

    Gardenia yellow is globally the most valuable spice and food color. It is generally a mixture of water-soluble carotenoid glycosyl esters which consist of crocetin bis(gentiobiosyl) ester as the main component. Crocetin is a natural carotenoid dicarboxylic acid that may be a candidate drug for pharmaceutical development, however, it is either present in trace amounts or is absent in natural gardenia yellow products. We here propose that crocetin produced by alkaline hydrolysis can be used to qualitatively evaluate gardenia yellow products using an ultra high performance liquid chromatographic assay. A useful and efficient isolation technique for isolating high-purity crocetin from gardenia yellow using high-speed countercurrent chromatography is described. High-speed countercurrent chromatographic fractionation followed by an ultra high performance liquid chromatographic assay showed that trans-crocetin is easily converted to about 15% cis-crocetin (85% trans-crocetin). Crocetin in gardenia yellow was quantitatively evaluated. Our approach is based on the hydrolysis process for converting crocetin glycosyl esters to crocetin before evaluation and isolation using the ultra high performance liquid chromatographic and high-speed countercurrent chromatographic methods. The combination of hydrolysis and chromatographic methods allows evaluation of the purity and quantity of crocetin in gardenia yellow.

  20. High-Mg# andesitic lavas of the Shisheisky Complex, Northern Kamchatka: implications for primitive calc-alkaline magmatism

    Science.gov (United States)

    Bryant, J. A.; Yogodzinski, G. M.; Churikova, T. G.

    2011-05-01

    Primitive arc magmatism and mantle wedge processes are investigated through a petrologic and geochemical study of high-Mg# (Mg/Mg + Fe > 0.65) basalts, basaltic andesites and andesites from the Kurile-Kamchatka subduction system. Primitive andesitic samples are from the Shisheisky Complex, a field of Quaternary-age, monogenetic cones located in the Aleutian-Kamchatka junction, north of Shiveluch Volcano, the northernmost active composite volcano in Kamchatka. The Shisheisky lavas have Mg# of 0.66-0.73 at intermediate SiO2 (54-58 wt%) with low CaO (3.0 wt%) and K2O (>1.0 wt%). Olivine phenocryst core compositions of Fo90 appear to be in equilibrium with whole-rock `melts', consistent with the sparsely phyric nature of the lavas. Compared to the Shisheisky andesites, primitive basalts from the region (Kuriles, Tolbachik, Kharchinsky) have higher CaO (>9.9 wt%) and CaO/Al2O3 (>0.60), and lower whole-rock Na2O (andesites. The absence of plagioclase phenocrysts from the primitive andesitic lavas contrasts the plagioclase-phyric basalts, indicating relatively high pre-eruptive water contents for the primitive andesitic magmas compared to basalts. Estimated temperature and water contents for primitive basaltic andesites and andesites are 984-1,143°C and 4-7 wt% H2O. For primitive basalts they are 1,149-1,227°C and 2 wt% H2O. Petrographic and mineral compositions suggest that the primitive andesitic lavas were liquids in equilibrium with mantle peridotite and were not produced by mixing between basalts and felsic crustal melts, contamination by xenocrystic olivine, or crystal fractionation of basalt. Key geochemical features of the Shisheisky primitive lavas (high Ni/MgO, Na2O, Ni/Yb and Mg# at intermediate SiO2) combined with the location of the volcanic field above the edge of the subducting Pacific Plate support a genetic model that involves melting of eclogite or pyroxenite at or near the surface of the subducting plate, followed by interaction of that melt with

  1. Comparative characterization of protease activity in cultured spotted rose snapper juveniles (Lutjanus guttatus

    Directory of Open Access Journals (Sweden)

    Emyr Peña

    2015-09-01

    Full Text Available Partial characterizations of digestive proteases were studied in three life stages of spotted rose snapper: early (EJ, middle (MJ and late juvenile (LJ with corresponding average weights of 21.3 ± 2.6 g (3 months after hatching, MAH, 190 ± 4.4 g (7 MAH, and 400 ± 11.5 g (12 MAH. At sampling points, the digestive tract was dissected into the stomach (St, pyloric caeca (PC, and the intestine in three sections (proximal (PI, middle (MI and distal intestine (DI. The effect of pH and temperature and specific inhibitors were evaluated for acid and alkaline proteases. Total acid and alkaline protease activity showed a tendency to increase with juvenile life stage of fish while trypsin activity decreased. Differences were found in acid and alkaline protease activities at different pH and temperatures during juvenile stages. Pepstatin A inhibited total activity in the stomach extract in all juvenile stages. Activity in total alkaline protease inhibition was significantly higher in EJ using TLCK, PMSF, SBTI, Phen and Ovo than in MJ and LJ, while no significant differences were found with TPCK inhibition. Therefore increases in protease activities with fish growth through juvenile stages in which a substitution or diversification in the type of alkaline enzymes exist. These results lead a better comprehension of changes in digestive potential of Lutjanidae fish.

  2. Purification and characterization of a protease produced by Bacillus megaterium RRM2: application in detergent and dehairing industries.

    Science.gov (United States)

    Rajkumar, Renganathan; Jayappriyan, Kothilmozhian Ranishree; Rengasamy, Ramasamy

    2011-12-01

    An alkaline serine protease produced by Bacillus megaterium RRM2 isolated from the red alga, Kappaphycus alvarezii (Doty) Doty ex Silva was studied for the first time and the same analyzed for the production of protease in the present study. Identification of the bacterium was done on the basis of both biochemical analysis and by 16S rDNA sequence analysis. The extracellular protease obtained from B. megaterium RRM2 was purified by a three-step process involving ammonium sulphate precipitation, gel filtration (Sephadex G100) and Q-Sepharose column chromatography. The purity was found to be 30.6-fold with a specific activity of 3591.5 U/mg protein with a molecular weight of 27 kDa. The metal ions Ca(2+), Mg(2+), K(+) and Na(+) marginally enhanced the activity of the purified enzyme while Hg(2+), Cu(2+), Fe(2+), CO(2+) and Zn(2+), had reduced the activity. The enzyme was found to be active in the pH range of 9.0-10.0 and remained active up to 60 °C. Phenyl Methyl Sulfonyl Fluoride (PMSF) inhibited the enzyme activity, thus, confirming that this enzyme is an alkaline serine protease. Likewise, DTT also inhibited the enzyme thus confirming the disulfide nature of the enzyme. The enzyme exhibited a high degree of tolerance to Sodium Dodecyl Sulphate (SDS). The partially purified protease when used as an additive in the commercial detergents was found to be a suitable source for washing clothes especially those stained with blood. Further, it showed good dehairing activity within a short duration in goat skin without affecting its collagen component.

  3. Characterizing proteases in an Antarctic Janthinobacterium sp. isolate:Evidence of a protease horizontal gene transfer event

    Institute of Scientific and Technical Information of China (English)

    Cecilia Martinez-Rosales; Juan Jos Marizcurrena; Andrs Iriarte; Natalia Fullana; Hctor Musto; Susana Castro-Sowinski

    2015-01-01

    We report the isolation of a cold-adapted bacterium belonging to the genus Janthinobacterium (named AU11), from a water sample collected in Lake Uruguay (King George Island, South Shetlands). AU11 (growth between 4°C and 30°C) produces a single cold-active extracellular protease (ExPAU11), differentially expressed at low temperature. ExPAU11 was identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS) as an alkaline metallo-protease (70% coverage with an extracellular protease of Janthinobacterium sp. PI12), and by protease-inhibitor screening identified as a serine-protease. To the best of our knowledge this is the first experimental evidence of a cold-active extracellular protease produced by Janthinobacterium. Furthermore, we identified a serine-protease gene (named JSP8A) showing 60% identity (98%query coverage) to subtilisin peptidases belonging to the S8 family (S8A subfamily) of many cyanobacteria. A phylogenetic analysis of the JSP8A protease, along with related bacterial protein sequences, confirms that JSP8A clusters with S8A subtilisin sequences from different cyanobacteria, and is clearly separated from S8A bacterial sequences of other phyla (including its own). An analysis of the genomic organization around JSP8A suggests that this protease gene was acquired in an event that duplicated a racemase gene involved in transforming L- to D-amino acids. Our results suggest that AU11 probably acquired this subtilisin-like protease gene by horizontal gene transfer (HGT) from a cyanobacterium. We discuss the relevance of a bacterial protease-HGT in the Antarctic environment in light of this hypothesis.

  4. High-resolution Imaging of pH in Alkaline Sediments and Water Based on a New Rapid Response Fluorescent Planar Optode.

    Science.gov (United States)

    Han, Chao; Yao, Lei; Xu, Di; Xie, Xianchuan; Zhang, Chaosheng

    2016-05-20

    A new dual-lumophore optical sensor combined with a robust RGB referencing method was developed for two-dimensional (2D) pH imaging in alkaline sediments and water. The pH sensor film consisted of a proton-permeable polymer (PVC) in which two dyes with different pH sensitivities and emission colors: (1) chloro phenyl imino propenyl aniline (CPIPA) and (2) the coumarin dye Macrolex(®) fluorescence yellow 10 GN (MFY-10 GN) were entrapped. Calibration experiments revealed the typical sigmoid function and temperature dependencies. This sensor featured high sensitivity and fast response over the alkaline working ranges from pH 7.5 to pH 10.5. Cross-sensitivity towards ionic strength (IS) was found to be negligible for freshwater when IS water associated with the photosynthesis of Vallisneria spiral species was also presented, suggesting that the sensor held great promise for the field applications.

  5. Novel fungal protease.

    NARCIS (Netherlands)

    Buxton, F.; Jarai, G.; Visser, J.

    1994-01-01

    The present invention concerns a novel DNA sequence coding for an Aspergillus serine protease of the subtilisin-type, an Aspergillus serine protease of the subtilisin-type per se and a method for the preparation thereof. The invention further concerns a novel Aspergillus mutant strain defective in a

  6. Methods of use of calcium hexa aluminate refractory linings and/or chemical barriers in high alkali or alkaline environments

    Science.gov (United States)

    McGowan, Kenneth A; Cullen, Robert M; Keiser, James R; Hemrick, James G; Meisner, Roberta A

    2013-10-22

    A method for improving the insulating character/and or penetration resistance of a liner in contact with at least one of an alkali and/or alkaline environments is provided. The method comprises lining a surface that is subject to wear by an alkali environment and/or an alkaline environment with a refractory composition comprising a refractory aggregate consisting essentially of a calcium hexa aluminate clinker having the formula CA.sub.6, wherein C is equal to calcium oxide, wherein A is equal to aluminum oxide, and wherein the hexa aluminate clinker has from zero to less than about fifty weight percent C.sub.12A.sub.7, and wherein greater than 98 weight percent of the calcium hexa aluminate clinker having a particle size ranging from -20 microns to +3 millimeters, for forming a liner of the surface. This method improves the insulating character/and or penetration resistance of the liner.

  7. Hollow raspberry-like PdAg alloy nanospheres: High electrocatalytic activity for ethanol oxidation in alkaline media

    Science.gov (United States)

    Peng, Cheng; Hu, Yongli; Liu, Mingrui; Zheng, Yixiong

    2015-03-01

    Palladium-silver (PdAg) alloy nanospheres with unique structure were prepared using a one-pot procedure based on the galvanic replacement reaction. Their electrocatalytic activity for ethanol oxidation in alkaline media was evaluated. The morphology and crystal structure of the samples were characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM) and X-ray diffraction (XRD). Electrochemical characterization techniques, including cyclic voltammetry (CV) and chronoamperometry (CA) measurements were used to analyze the electrochemical performance of the PdAg alloy nanospheres. The SEM and TEM images showed that the PdAg alloy nanospheres exhibit a hierarchical nanostructure with hollow interiors and porous walls. Compared to the commercial Pd/C catalyst, the as-prepared PdAg alloy nanospheres exhibit superior electrocatalytic activity and stability towards ethanol electro-oxidation in alkaline media, showing its potential as a new non-Pt electro-catalyst for direct alcohol fuel cells (DAFCs).

  8. High content image-based screening of a protease inhibitor library reveals compounds broadly active against Rift Valley fever virus and other highly pathogenic RNA viruses.

    Directory of Open Access Journals (Sweden)

    Rajini Mudhasani

    2014-08-01

    Full Text Available High content image-based screening was developed as an approach to test a protease inhibitor small molecule library for antiviral activity against Rift Valley fever virus (RVFV and to determine their mechanism of action. RVFV is the causative agent of severe disease of humans and animals throughout Africa and the Arabian Peninsula. Of the 849 compounds screened, 34 compounds exhibited ≥ 50% inhibition against RVFV. All of the hit compounds could be classified into 4 distinct groups based on their unique chemical backbone. Some of the compounds also showed broad antiviral activity against several highly pathogenic RNA viruses including Ebola, Marburg, Venezuela equine encephalitis, and Lassa viruses. Four hit compounds (C795-0925, D011-2120, F694-1532 and G202-0362, which were most active against RVFV and showed broad-spectrum antiviral activity, were selected for further evaluation for their cytotoxicity, dose response profile, and mode of action using classical virological methods and high-content imaging analysis. Time-of-addition assays in RVFV infections suggested that D011-2120 and G202-0362 targeted virus egress, while C795-0925 and F694-1532 inhibited virus replication. We showed that D011-2120 exhibited its antiviral effects by blocking microtubule polymerization, thereby disrupting the Golgi complex and inhibiting viral trafficking to the plasma membrane during virus egress. While G202-0362 also affected virus egress, it appears to do so by a different mechanism, namely by blocking virus budding from the trans Golgi. F694-1532 inhibited viral replication, but also appeared to inhibit overall cellular gene expression. However, G202-0362 and C795-0925 did not alter any of the morphological features that we examined and thus may prove to be good candidates for antiviral drug development. Overall this work demonstrates that high-content image analysis can be used to screen chemical libraries for new antivirals and to determine their

  9. Molecular Cloning and Optimization for High Level Expression of Cold-Adapted Serine Protease from Antarctic Yeast Glaciozyma antarctica PI12

    Directory of Open Access Journals (Sweden)

    Norsyuhada Alias

    2014-01-01

    Full Text Available Psychrophilic basidiomycete yeast, Glaciozyma antarctica strain PI12, was shown to be a protease-producer. Isolation of the PI12 protease gene from genomic and mRNA sequences allowed determination of 19 exons and 18 introns. Full-length cDNA of PI12 protease gene was amplified by rapid amplification of cDNA ends (RACE strategy with an open reading frame (ORF of 2892 bp, coded for 963 amino acids. PI12 protease showed low homology with the subtilisin-like protease from fungus Rhodosporidium toruloides (42% identity and no homology to other psychrophilic proteases. The gene encoding mature PI12 protease was cloned into Pichia pastoris expression vector, pPIC9, and positioned under the induction of methanol-alcohol oxidase (AOX promoter. The recombinant PI12 protease was efficiently secreted into the culture medium driven by the Saccharomyces cerevisiae α-factor signal sequence. The highest protease production (28.3 U/ml was obtained from P. pastoris GS115 host (GpPro2 at 20°C after 72 hours of postinduction time with 0.5% (v/v of methanol inducer. The expressed protein was detected by SDS-PAGE and activity staining with a molecular weight of 99 kDa.

  10. Comparison of in vitro behavior of as-sprayed, alkaline-treated and collagen-treated bioceramic coatings obtained by high velocity oxy-fuel spray

    Energy Technology Data Exchange (ETDEWEB)

    Melero, H., E-mail: hortensia.melero.correas@gmail.com [Thermal Spray Centre, Universitat de Barcelona, Martí i Franqués, 1, 08028 Barcelona (Spain); Garcia-Giralt, N. [URFOA, IMIM (Institut Hospital del Mar d’Investigacions Mèdiques), RETICEF, Doctor Aiguader, 80, 08003 Barcelona (Spain); Fernández, J. [Thermal Spray Centre, Universitat de Barcelona, Martí i Franqués, 1, 08028 Barcelona (Spain); Díez-Pérez, A. [URFOA, IMIM (Institut Hospital del Mar d’Investigacions Mèdiques), RETICEF, Doctor Aiguader, 80, 08003 Barcelona (Spain); Servei de Medicina Interna, Hospital del Mar, Barcelona (Spain); Guilemany, J.M. [Thermal Spray Centre, Universitat de Barcelona, Martí i Franqués, 1, 08028 Barcelona (Spain)

    2014-07-01

    Hydroxyapatite (HAp)–TiO{sub 2} samples obtained using high velocity oxy-fuel spray (HVOF), that had previously shown excellent mechanical behaviour, were innovatively surface treated in order to improve their biological performance. The chosen treatments were an alkaline treatment to increase –OH radicals density on the surface (especially on TiO{sub 2} zones), and a collagen treatment to bond collagen fibrils to the –OH radicals present in hydroxyapatite. These coatings were analysed using scanning electron microscopy, energy-dispersive X-ray spectroscopy and infrared spectroscopy, and tested for human osteoblast biocompatibility and functionality. In the case of the alkaline treatment, although the –OH radicals density did not increase compared to the as-sprayed coatings, a nanostructured layer of sodium hydroxycarbonate precipitated on the surface, thus improving biological behaviour due to the nanoroughness effect. For the collagen-treated samples, collagen fibrils appeared well-adhered to the surface, and in vitro cell culture tests showed that these surfaces were much more conducive to cell adhesion and differentiation than the as-sprayed and alkaline-treated samples. These results pointed to collagen treatment as a very promising method to improve bioactivity of HAp–TiO{sub 2} thermal-sprayed coatings.

  11. The evolution of Neoproterozoic magmatism in Southernmost Brazil: shoshonitic, high-K tholeiitic and silica-saturated, sodic alkaline volcanism in post-collisional basins

    Directory of Open Access Journals (Sweden)

    Sommer Carlos A.

    2006-01-01

    Full Text Available The Neoproterozoic shoshonitic and mildly alkaline bimodal volcanism of Southernmost Brazil is represented by rock assemblages associated to sedimentary successions, deposited in strike-slip basins formed at the post-collisional stages of the Brasilian/Pan-African orogenic cycle. The best-preserved volcano sedimentary associations occur in the Camaquã and Campo Alegre Basins, respectively in the Sul-riograndense and Catarinense Shields and are outside the main shear belts or overlying the unaffected basement areas. These basins are characterized by alternation of volcanic cycles and siliciclastic sedimentation developed dominantly on a continental setting under subaerial conditions. This volcanism and the coeval plutonism evolved from high-K tholeiitic and calc-alkaline to shoshonitic and ended with a silica-saturated sodic alkaline magmatism, and its evolution were developed during at least 60 Ma. The compositional variation and evolution of post-collisional magmatism in southern Brazil are interpreted as the result mainly of melting of a heterogeneous mantle source, which includes garnet-phlogopite-bearing peridotites, veined-peridotites with abundant hydrated phases, such as amphibole, apatite and phlogopite, and eventually with the addition of an asthenospheric component. The subduction-related metasomatic character of post-collisional magmatism mantle sources in southern Brazil is put in evidence by Nb-negative anomalies and isotope features typical of EM1 sources.

  12. A General Silica-Templating Synthesis of Alkaline Mesoporous Carbon Catalysts for Highly Efficient H2S Oxidation at Room Temperature.

    Science.gov (United States)

    Zhang, Zixiao; Jiang, Wuyou; Long, Donghui; Wang, Jitong; Qiao, Wenming; Ling, Licheng

    2017-01-25

    A general synthesis of alkaline mesoporous carbons (AMCs) is developed based on a simplified silica-templating method for room-temperature catalytic oxidation of H2S. The key to the success relies on dissolving the silica templates to create the interconnected mesoporous structure as well as leaving parts of the alkaline products in the pores; both of them are prerequisites for H2S oxidation. By adjusting the alkaline etching degree and organic/inorganic ratio, the porosity and basicity of the AMC could be simultaneously tuned, allowing the AMCs direct use for H2S catalytic oxidation with an unprecedented removal capacities of 4.49 ± 0.12 g/g. Such excellent catalytic performance should be attributed to the developed pore structure that stores the product sulfur and the strong basicity that promotes the dissociation of H2S into HS(-) ions. Moreover, this simplified silica-templating method could be easily extended to the preparation of various silica templated mesoporous carbon catalysts. All these AMCs demonstrate a successful combination of low cost with high performance, which may well be the answer for the technical development of industrial H2S removal.

  13. Recombinant Deg/HtrA proteases from Synechocystis sp. PCC 6803 differ in substrate specificity, biochemical characteristics and mechanism

    Science.gov (United States)

    Huesgen, Pitter F.; Miranda, Helder; Lam, XuanTam; Perthold, Manuela; Schuhmann, Holger; Adamska, Iwona; Funk, Christiane

    2011-01-01

    Cyanobacteria require efficient protein-quality-control mechanisms to survive under dynamic, often stressful, environmental conditions. It was reported that three serine proteases, HtrA (high temperature requirement A), HhoA (HtrA homologue A) and HhoB (HtrA homologue B), are important for survival of Synechocystis sp. PCC 6803 under high light and temperature stresses and might have redundant physiological functions. In the present paper, we show that all three proteases can degrade unfolded model substrates, but differ with respect to cleavage sites, temperature and pH optima. For recombinant HhoA, and to a lesser extent for HtrA, we observed an interesting shift in the pH optimum from slightly acidic to alkaline in the presence of Mg2+ and Ca2+ ions. All three proteases formed different homo-oligomeric complexes with and without substrate, implying mechanistic differences in comparison with each other and with the well-studied Escherichia coli orthologues DegP (degradation of periplasmic proteins P) and DegS. Deletion of the PDZ domain decreased, but did not abolish, the proteolytic activity of all three proteases, and prevented substrate-induced formation of complexes higher than trimers by HtrA and HhoA. In summary, biochemical characterization of HtrA, HhoA and HhoB lays the foundation for a better understanding of their overlapping, but not completely redundant, stress-resistance functions in Synechocystis sp. PCC 6803. PMID:21332448

  14. Screening and characterization of protease producing actinomycetes from marine saltern.

    Science.gov (United States)

    Suthindhiran, Krish; Jayasri, Mangalam Achuthananda; Dipali, Dipa; Prasar, Apurva

    2014-10-01

    In the course of systematic screening program for bioactive actinomycetes, an alkaline protease producing halophilic strain Actinopolyspora sp. VITSDK2 was isolated from marine saltern, Southern India. The strain was identified as Actinopolyspora based on its phenotypic and phylogenetic characters. The protease was partially purified using ammonium sulfate precipitation and subsequently by DEAE cellulose column chromatography. The enzyme was further purified using HPLC and the molecular weight was found to be 22 kDa as determined by SDS-PAGE analysis. The purified protease exhibited pH stability in a wide range of 4-12 with optimum at 10.0. The enzyme was found to be stable between 25 and 80 °C and displayed a maximum activity at 60 °C. The enzyme activity was increased marginally in presence of Mn(2+) , Mg(2+) , and Ca(2+) and decreased in presence of Cu(2+) . PMSF and DFP completely inhibited the activity suggesting it belongs to serine protease. Further, the proteolytic activity was abolished in presence of N-tosyl-L-lysine chloromethyl ketone suggesting this might be chymotrypsin-like serine protease. The protease was 96% active when kept for 10 days at room temperature. The results indicate that the enzyme belong to chymotrypsin-like serine protease exhibiting both pH and thermostability, which can be used for various applications in industries. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Increasing the alkaline protease activity of Bacillus cereus and ...

    African Journals Online (AJOL)

    User

    2011-05-09

    May 9, 2011 ... activities of B. polymyxa and B. cereus were measured at the death phase of the growth curve and at the start of the ... in glucose synthetic medium at 30°C with 100 rpm aeration speed for 144 h. .... surface, gray to light brown.

  16. Production of alkaline proteases by alkalophilic Bacillus subtilis ...

    African Journals Online (AJOL)

    Tuoyo Aghomotsegin

    2016-11-23

    Nov 23, 2016 ... Bacillus subtilis during recycling animal and plant wastes. Amira Hassan .... collected from farms in sterile plastic bags (Sonia Sethi et al.,. 2012). ... Effect of different waste concentrations on enzyme production. To test the best ...

  17. Comparative Study on the Protease Inhibitors from Fish Eggs

    Institute of Scientific and Technical Information of China (English)

    Ustadi; K.Y.Kim; S.M.Kim

    2005-01-01

    The protease inhibitor was purified from five different fish eggs. The molecular weights of Pacific herring, chum salmon, pond smelt, glassfish, and Alaska pollock egg protease inhibitors were 120, 89, 84.5, 17, and 16.8kDa, respectively. The specific inhibitory activity of glassfish egg protease inhibitor was the highest followed by those of Pacific herring and Alaska pollock in order. The specific inhibitory activity and purity of glassfish egg protease inhibitor were 19.70 U mg-1 protein and 164.70 folds of purification, respectively. Glassfish egg protease inhibitor was reasonably stable at 50 - 65 ℃ and pH 8,which was more stable at high temperature and pH than protease inhibitors from the other fish species. Glassfish egg protease inhibitor was noncompetitive with inhibitor constant (Ki) of 4.44 nmol L-1.

  18. C-5-Modified Tetrahydropyrano-Tetrahydofuran-Derived Protease Inhibitors (PIs) Exert Potent Inhibition of the Replication of HIV-1 Variants Highly Resistant to Various PIs, including Darunavir

    Science.gov (United States)

    Aoki, Manabu; Hayashi, Hironori; Yedidi, Ravikiran S.; Martyr, Cuthbert D.; Takamatsu, Yuki; Aoki-Ogata, Hiromi; Nakamura, Teruya; Nakata, Hirotomo; Das, Debananda; Yamagata, Yuriko; Ghosh, Arun K.

    2015-01-01

    ABSTRACT We identified three nonpeptidic HIV-1 protease inhibitors (PIs), GRL-015, -085, and -097, containing tetrahydropyrano-tetrahydrofuran (Tp-THF) with a C-5 hydroxyl. The three compounds were potent against a wild-type laboratory HIV-1 strain (HIV-1WT), with 50% effective concentrations (EC50s) of 3.0 to 49 nM, and exhibited minimal cytotoxicity, with 50% cytotoxic concentrations (CC50) for GRL-015, -085, and -097 of 80, >100, and >100 μM, respectively. All the three compounds potently inhibited the replication of highly PI-resistant HIV-1 variants selected with each of the currently available PIs and recombinant clinical HIV-1 isolates obtained from patients harboring multidrug-resistant HIV-1 variants (HIVMDR). Importantly, darunavir (DRV) was >1,000 times less active against a highly DRV-resistant HIV-1 variant (HIV-1DRVRP51); the three compounds remained active against HIV-1DRVRP51 with only a 6.8- to 68-fold reduction. Moreover, the emergence of HIV-1 variants resistant to the three compounds was considerably delayed compared to the case of DRV. In particular, HIV-1 variants resistant to GRL-085 and -097 did not emerge even when two different highly DRV-resistant HIV-1 variants were used as a starting population. In the structural analyses, Tp-THF of GRL-015, -085, and -097 showed strong hydrogen bond interactions with the backbone atoms of active-site amino acid residues (Asp29 and Asp30) of HIV-1 protease. A strong hydrogen bonding formation between the hydroxyl moiety of Tp-THF and a carbonyl oxygen atom of Gly48 was newly identified. The present findings indicate that the three compounds warrant further study as possible therapeutic agents for treating individuals harboring wild-type HIV and/or HIVMDR. IMPORTANCE Darunavir (DRV) inhibits the replication of most existing multidrug-resistant HIV-1 strains and has a high genetic barrier. However, the emergence of highly DRV-resistant HIV-1 strains (HIVDRVR) has recently been observed in vivo and in

  19. Highly efficient infectious cell culture of three HCV genotype 2b strains and sensitivity to lead protease, NS5A, and polymerase inhibitors

    DEFF Research Database (Denmark)

    Ramirez, Santseharay; Li, Yi-Ping; Brun Jensen, Sanne;

    2014-01-01

    , we succeeded in generating DH8, J8, and DH10 viruses with authentic sequences in the regions targeted by lead direct acting antivirals. NS5B inhibitors Sofosbuvir, Mericitabine, and BI207127 had activity against 1a (strain TN), 2a (strains JFH1 and J6), and the 2b strains, whereas VX-222...... systems can be established by using consensus clones with defined mutations. Lead protease and NS5A inhibitors, as well as polymerase inhibitors Sofosbuvir, Mericitabine, and BI207127, show cross-activity against full-length 1a, 2a, and 2b viruses, but important sensitivity differences exist......Hepatitis C virus (HCV) is a genetically diverse virus with multiple genotypes exhibiting remarkable differences, particularly in drug susceptibility. Drug and vaccine development will benefit from high-titer HCV cultures mimicking the complete viral life cycle, but such systems only exist...

  20. Evaluation of high dietary inclusion of distillers dried grains with solubles and supplementation of protease and xylanase in the diets of broiler chickens under necrotic enteritis challenge.

    Science.gov (United States)

    Barekatain, M R; Antipatis, C; Rodgers, N; Walkden-Brown, S W; Iji, P A; Choct, M

    2013-06-01

    A 2 × 2 × 2 factorial experiment was conducted to investigate the effect of a high level of sorghum distillers dried grains with solubles (DDGS; 20%), with or without a combination of protease and xylanase in broiler chickens, under a necrotic enteritis disease challenge. A total of 576 male broiler chicks were randomly assigned to 8 experimental treatments, each replicated 6 times, with 12 birds per replicate for 35 d. Oral inoculation of the challenged group with Eimeria spp. occurred on d 9, followed by 3 consecutive inoculations of Clostridium perfringens from d 14 through 16. The disease challenge and DDGS inclusion significantly (P enteritis-related lesions (d 17) were more severe (P enteritis. Supplementation of enzymes did not reveal significant mitigation effect in infected birds but helped the birds fed DDGS to maintain feed intake and BW gain.

  1. Pulmonary Proteases in the Cystic Fibrosis Lung Induce Interleukin 8 Expression from Bronchial Epithelial Cells via a Heme/Meprin/Epidermal Growth Factor Receptor/Toll-like Receptor Pathway.

    LENUS (Irish Health Repository)

    Cosgrove, Sonya

    2011-03-04

    A high intrapulmonary protease burden is characteristic of cystic fibrosis (CF), and the resulting dysregulation of the protease\\/anti-protease balance has serious implications for inflammation in the CF lung. Because of this inflammation, micro-bleeds can occur releasing hemoglobin into the lung. The aim of this study was to investigate the effect of the protease-rich environment of the CF lung on human hemoglobin and to assess the proinflammatory effect of heme on CF bronchial epithelium. Here, we show that the Pseudomonas proteases (Pseudomonas elastase and alkaline protease) and the neutrophil proteases (neutrophil elastase (NE) and proteinase-3) are capable of almost complete degradation of hemoglobin in vitro but that NE is the predominant protease that cleaves hemoglobin in vivo in CF bronchoalveolar lavage fluid. One of the effects of this is the release of heme, and in this study we show that heme stimulates IL-8 and IL-10 protein production from ΔF508 CFBE41o(-) bronchial epithelial cells. In addition, heme-induced IL-8 expression utilizes a novel pathway involving meprin, EGF receptor, and MyD88. Meprin levels are elevated in CF cell lines and bronchial brushings, thus adding to the proinflammatory milieu. Interestingly, α(1)-antitrypsin, in addition to its ability to neutralize NE and protease-3, can also bind heme and neutralize heme-induced IL-8 from CFBE41o(-) cells. This study illustrates the proinflammatory effects of micro-bleeds in the CF lung, the process by which this occurs, and a potential therapeutic intervention.

  2. Pulmonary proteases in the cystic fibrosis lung induce interleukin 8 expression from bronchial epithelial cells via a heme/meprin/epidermal growth factor receptor/Toll-like receptor pathway.

    LENUS (Irish Health Repository)

    Cosgrove, Sonya

    2012-02-01

    A high intrapulmonary protease burden is characteristic of cystic fibrosis (CF), and the resulting dysregulation of the protease\\/anti-protease balance has serious implications for inflammation in the CF lung. Because of this inflammation, micro-bleeds can occur releasing hemoglobin into the lung. The aim of this study was to investigate the effect of the protease-rich environment of the CF lung on human hemoglobin and to assess the proinflammatory effect of heme on CF bronchial epithelium. Here, we show that the Pseudomonas proteases (Pseudomonas elastase and alkaline protease) and the neutrophil proteases (neutrophil elastase (NE) and proteinase-3) are capable of almost complete degradation of hemoglobin in vitro but that NE is the predominant protease that cleaves hemoglobin in vivo in CF bronchoalveolar lavage fluid. One of the effects of this is the release of heme, and in this study we show that heme stimulates IL-8 and IL-10 protein production from DeltaF508 CFBE41o(-) bronchial epithelial cells. In addition, heme-induced IL-8 expression utilizes a novel pathway involving meprin, EGF receptor, and MyD88. Meprin levels are elevated in CF cell lines and bronchial brushings, thus adding to the proinflammatory milieu. Interestingly, alpha(1)-antitrypsin, in addition to its ability to neutralize NE and protease-3, can also bind heme and neutralize heme-induced IL-8 from CFBE41o(-) cells. This study illustrates the proinflammatory effects of micro-bleeds in the CF lung, the process by which this occurs, and a potential therapeutic intervention.

  3. MALT1 protease: equilibrating immunity versus tolerance

    OpenAIRE

    Bertossi, Arianna; Krappmann, Daniel

    2014-01-01

    MALT1 paracaspase links signaling cascades emanating from adaptive or innate immune receptors to the canonical NF-κB pathway. Now, Jaworski et al (2014) investigate the physiological role of MALT1 protease activity in mice. Besides the expected requirement of MALT1 activity for immune activation, the study unveils a novel function for MALT1 activity for the development of peripheral tolerance. Thus, MALT1 protease can act immunogenic or tolerogenic, and this interplay will be highly relevant ...

  4. Serine protease espP subtype alpha, but not beta or gamma, of Shiga toxin-producing Escherichia coli is associated with highly pathogenic serogroups.

    Science.gov (United States)

    Khan, Abdul Basit; Naim, Asma; Orth, Dorothea; Grif, Katharina; Mohsin, Mashkoor; Prager, Rita; Dierich, Manfred P; Würzner, Reinhard

    2009-04-01

    Besides Shiga toxins (Stx), Stx-producing Escherichia coli (STEC) harbour several other putative virulence factors, including the serine protease EspP. We have investigated 214 STEC strains from Austria belonging to 61 different serotypes from humans, animals, and food for the presence of this serine protease gene and have determined the espP subtypes and their association with clinical outcome. espP was detected in 121 (57%) out of 214 strains. Sixty-five of 68 strains (96%) of non-sorbitol-fermenting (NSF) O157:H7/NM (NM, non-motile) were positive for espP, while none of 8 SF E. coli O157:NM isolates contained this gene. All 9 strains of serotype O145:NM and 17 of 21 strains (81%) of serotype O26:H11/NM were positive for espP. Nineteen STEC serogroups including O103 and O111 serogroups--considered to be highly pathogenic--were completely negative for espP. Only 5 of 12 strains isolated from patients suffering from haemolytic uraemic syndrome (HUS) were espP-positive (all serogroup NSF O157) as well as 28 of 39 strains from patients with bloody diarrhoea, 40 of 63 strains from patients with non-bloody diarrhoea, and 15 of 19 strains from asymptomatic patients. In O157:H7/NM, O26:H11/NM, and O145:NM only espP subtype alpha was found, whereas in most of the other non-O157 serogroups, subtypes beta and gamma were found. Subtype delta was not detected in our strain collection. Regarding the espP subtypes, only subtype alpha, but not beta and gamma, were found in HUS patients. Moreover, we could demonstrate that espP, and in particular subtype alpha, is associated with highly pathogenic serogroups.

  5. High performance nano-Ni/Graphite electrode for electro-oxidation in direct alkaline ethanol fuel cells

    Science.gov (United States)

    Soliman, Ahmed B.; Abdel-Samad, Hesham S.; Abdel Rehim, Sayed S.; Ahmed, Mohamed A.; Hassan, Hamdy H.

    2016-09-01

    Ni/Graphite electrocatalysts (Ni/G) are successfully prepared through electrodeposition of Ni from acidic (pH = 0.8) and feebly acidic (pH = 5.5) aqueous Ni (II) baths. The efficiencies of such electrodes are investigated as anodes for direct alkaline ethanol fuel cells through their ethanol electrooxidation cyclic voltammetric (CV) response in alkaline medium. A direct proportionality between the amount of the electrodeposited Ni and its CV response is found. The amounts of the deposited Ni from the two baths are recorded using the Electrochemical Quartz Crystal Microbalance (eQCM). The Ni/G electrodes prepared from the feebly acidic bath show a higher electrocatalytic response than those prepared from the acidic bath. Surface morphology of the Ni particles electrodeposited from feebly acidic bath appears in a nano-scale dimension. Various electrochemical experiments are conducted to confirm that the Ni/G ethanol electrooxidation CV response greatly depends on the pH rather than nickel ion concentration of the deposition bath. The eQCM technique is used to detect the crystalline phases of nickel as α-Ni(OH)2/γ-NiOOH and β-Ni(OH)2/β-NiOOH and their in-situ inter-transformations during the potentiodynamic polarization.

  6. Secreted fungal aspartic proteases: A review.

    Science.gov (United States)

    Mandujano-González, Virginia; Villa-Tanaca, Lourdes; Anducho-Reyes, Miguel Angel; Mercado-Flores, Yuridia

    2016-01-01

    The aspartic proteases, also called aspartyl and aspartate proteases or acid proteases (E.C.3.4.23), belong to the endopeptidase family and are characterized by the conserved sequence Asp-Gly-Thr at the active site. These enzymes are found in a wide variety of microorganisms in which they perform important functions related to nutrition and pathogenesis. In addition, their high activity and stability at acid pH make them attractive for industrial application in the food industry; specifically, they are used as milk-coagulating agents in cheese production or serve to improve the taste of some foods. This review presents an analysis of the characteristics and properties of secreted microbial aspartic proteases and their potential for commercial application. Copyright © 2016 Asociación Española de Micología. Published by Elsevier Espana. All rights reserved.

  7. Bacterial proteases and virulence

    DEFF Research Database (Denmark)

    Frees, Dorte; Brøndsted, Lone; Ingmer, Hanne

    2013-01-01

    Bacterial pathogens rely on proteolysis for variety of purposes during the infection process. In the cytosol, the main proteolytic players are the conserved Clp and Lon proteases that directly contribute to virulence through the timely degradation of virulence regulators and indirectly by providing....... These extracellular proteases are activated in complex cascades involving auto-processing and proteolytic maturation. Thus, proteolysis has been adopted by bacterial pathogens at multiple levels to ensure the success of the pathogen in contact with the human host....

  8. Gag mutations strongly contribute to HIV-1 resistance to protease inhibitors in highly drug-experienced patients besides compensating for fitness loss.

    Directory of Open Access Journals (Sweden)

    Elisabeth Dam

    2009-03-01

    Full Text Available Human immunodeficiency virus type 1 (HIV-1 resistance to protease inhibitors (PI results from mutations in the viral protease (PR that reduce PI binding but also decrease viral replicative capacity (RC. Additional mutations compensating for the RC loss subsequently accumulate within PR and in Gag substrate cleavage sites. We examined the respective contribution of mutations in PR and Gag to PI resistance and RC and their interdependence using a panel of HIV-1 molecular clones carrying different sequences from six patients who had failed multiple lines of treatment. Mutations in Gag strongly and directly contributed to PI resistance besides compensating for fitness loss. This effect was essentially carried by the C-terminal region of Gag (containing NC-SP2-p6 with little or no contribution from MA, CA, and SP1. The effect of Gag on resistance depended on the presence of cleavage site mutations A431V or I437V in NC-SP2-p6 and correlated with processing of the NC/SP2 cleavage site. By contrast, reverting the A431V or I437V mutation in these highly evolved sequences had little effect on RC. Mutations in the NC-SP2-p6 region of Gag can be dually selected as compensatory and as direct PI resistance mutations, with cleavage at the NC-SP2 site behaving as a rate-limiting step in PI resistance. Further compensatory mutations render viral RC independent of the A431V or I437V mutations while their effect on resistance persists.

  9. Static and Dynamic Structure Factors with Account of the Ion Structure for High-temperature Alkali and Alkaline Earth Plasmas

    CERN Document Server

    Sadykova, S P; Tkachenko, I M

    2010-01-01

    The $e-e$, $e-i$, $i-i$ and charge-charge static structure factors are calculated for alkali and Be$^{2+}$ plasmas using the method described by Gregori et al. in \\cite{bibGreg2006}. The dynamic structure factors for alkali plasmas are calculated using the method of moments \\cite{bibAdam83}, \\cite{bibAdam93}. In both methods the screened Hellmann-Gurskii-Krasko potential, obtained on the basis of Bogolyubov's method, has been used taking into account not only the quantum-mechanical effects but also the ion structure \\cite{bib73}. PACS: 52.27.Aj (Alkali and alkaline earth plasmas, Static and dynamic structure factors), 52.25.Kn (Thermodynamics of plasmas), 52.38.Ph (X-ray scattering)

  10. Alkaline fuel cells applications

    Science.gov (United States)

    Kordesch, Karl; Hacker, Viktor; Gsellmann, Josef; Cifrain, Martin; Faleschini, Gottfried; Enzinger, Peter; Fankhauser, Robert; Ortner, Markus; Muhr, Michael; Aronson, Robert R.

    On the world-wide automobile market technical developments are increasingly determined by the dramatic restriction on emissions as well as the regimentation of fuel consumption by legislation. Therefore there is an increasing chance of a completely new technology breakthrough if it offers new opportunities, meeting the requirements of resource preservation and emission restrictions. Fuel cell technology offers the possibility to excel in today's motive power techniques in terms of environmental compatibility, consumer's profit, costs of maintenance and efficiency. The key question is economy. This will be decided by the costs of fuel cell systems if they are to be used as power generators for future electric vehicles. The alkaline hydrogen-air fuel cell system with circulating KOH electrolyte and low-cost catalysed carbon electrodes could be a promising alternative. Based on the experiences of Kordesch [K. Kordesch, Brennstoffbatterien, Springer, Wien, 1984, ISBN 3-387-81819-7; K. Kordesch, City car with H 2-air fuel cell and lead-battery, SAE Paper No. 719015, 6th IECEC, 1971], who operated a city car hybrid vehicle on public roads for 3 years in the early 1970s, improved air electrodes plus new variations of the bipolar stack assembly developed in Graz are investigated. Primary fuel choice will be a major issue until such time as cost-effective, on-board hydrogen storage is developed. Ammonia is an interesting option. The whole system, ammonia dissociator plus alkaline fuel cell (AFC), is characterised by a simple design and high efficiency.

  11. The total protein content, protein fractions and proteases activities of drone prepupae of Apis mellifera due to varrosis.

    Science.gov (United States)

    Zółtowska, Krystyna; Lipiński, Zbigniew; Dmitryjuk, Małgorzata

    2005-01-01

    The proteins level and activities of acid and alkaline proteases in whole body extracts of drone prepupae of Apis mellifera naturally infested with Varroa destructor were studied. The infested and a non-infested group did not differ significantly in their total protein content. However, some differences in protein profiles were found. A lack of three protein fractions of moderate and lower molecular weight in infested prepupae was noted. Moreover, some differences in the quantity of protein in most of the fractions were observed. The activity of acid proteases from infested prepupae was lower (p drone had higher activity of alkaline proteases than non-infested but this difference was not statisticaly significant.

  12. Preparation of Reducing Sugar Hydrolyzed from High-Lignin Coconut Coir Dust Pretreated by the Recycled Ionic Liquid [mmim][dmp] and Combination with Alkaline

    Directory of Open Access Journals (Sweden)

    Hanny Frans Sangian

    2015-03-01

    Full Text Available This study aims to produce reducing sugar hydrolyzed from substrate, coconut coir dust pretreated by recycled ionic liquid and its combination with alkaline. The 1H NMR and FTIR were performed to ver-ify the synthesized ionic liquid methylmethylimidazolium dimethyl phosphate ([mmim][dmp]. The structure of pretreated substrates was analyzed by XRD measurement. The used ionic liquid was recy-cled twice to re-employ for substrate pretreatment. The treated- and untreated-coconut coir dust were hydrolyzed into sugars using pure cellulase. The reaction, which called an enzymatic hydrolysis, was conducted at 60 °C, pH 3, for 48 h. The yields of sugar hydrolyzed from fresh IL-pretreated, 1R*IL-pretreated and 2R*IL-pretreated substrates were of 0.19, 0.15 and 0.15 g sugar / g cellu-lose+hemicellulose, respectively. Pretreatment with NaOH or the combination of NaOH+IL resulted in yields of reducing sugars of 0.25, 0.28 g/g, respectively. When alkaline combined with the recycled ionic liquids, NaOH+1R*IL, NaOH+2R*IL in the pretreatment, the yields of sugar were relatively similar to those obtained using alkaline followed by fresh ionic liquid. If the mixture enzymes, cellu-lase+xylanase, used to liberate sugars from fresh IL-pretreated, or recycled IL-pretreated substrates, the amount of sugar (concentration or yield increased slightly compared to that employing a single cel-lulase. These findings showed that recycled IL pretreatment of the high-lignin lignocellulose, coconut coir dust, is a new prospect for the economical manufacture of fermentable sugars and biofuel in the coming years. © 2015 BCREC UNDIP. All rights reservedReceived: 1st July 2014; Revised: 5th September 2014; Accepted: 5th September 2014 How to Cite: Sangian, H.F., Kristian, J., Rahma, S., Dewi, H., Puspasari, D., Agnesty, S., Gunawan, S., Widjaja, A. (2015. Preparation of Reducing Sugar Hydrolyzed from High-Lignin Coconut Coir Dust Pretreated by the Recycled Ionic Liquid [mmim

  13. Rice bifunctional phytocystatin is a dual modulator of legumain and papain-like proteases.

    Science.gov (United States)

    Christoff, Ana Paula; Passaia, Gisele; Salvati, Caroline; Alves-Ferreira, Márcio; Margis-Pinheiro, Marcia; Margis, Rogerio

    2016-09-01

    Phytocystatins are well-known inhibitors of C1A cysteine proteinases. However, previous research has revealed legumain (C13) protease inhibition via a carboxy-extended phytocystatin. Among the 12 phytocystatins genes in rice, OcXII is the only gene possessing this carboxy-terminal extension. The specific legumain inhibition activity was confirmed, in our work, using a recombinant OcXII harboring only the carboxy-terminal domain and this part did not exhibit any effect on papain-like activities. Meanwhile, rice plants silenced at the whole OcXII gene presented higher legumain and papain-like proteolytic activities, resulting in a faster initial seedling growth. However, when germinated under stressful alkaline conditions, OcXII-silenced plants exhibited impaired root formation and delayed shoot growth. Interestingly, the activity of OcXII promoter gene was detected in the rice seed scutellum region, and decreases with seedling growth. Seeds from these plants also exhibited slower growth at germination under ABA or alkaline conditions, while maintaining very high levels of OcXII transcriptional activation. This likely reinforces the proteolytic control necessary for seed germination and growth. In addition, increased legumain activity was detected in OcXII RNAi plants subjected to a fungal elicitor. Overall, the results of this study highlight the association of OcXII with not only plant development processes, but also with stress response pathways. The results of this study reinforce the bifunctional ability of carboxy-extended phytocystatins in regulating legumain proteases via its carboxy-extended domain and papain-like proteases by its amino-terminal domain.

  14. An alkaline element

    Energy Technology Data Exchange (ETDEWEB)

    Arita, T.; Murakami, K.; Okha, K.

    1983-04-28

    A cathode with a dual layer active mass is installed in the disk shaped alkaline silver and zinc element. The first layer, which is turned towards the anode, contains 85 parts Ag2O, 5 parts electrolytic MnO2 and 10 parts graphite. The second layer, which contacts the bottom of the element, contains 35 parts Ag2O, 60 parts electrolytic MnO2 and 5 parts graphite. The electrical capacity of the first and second layers is 60 and 40, respectively. The first layer may be discharged with a high current density and the second layer with less current density. The element has high characteristics with comparatively low cost.

  15. Sorption of water alkalinity and hardness from high-strength wastewater on bifunctional activated carbon: process optimization, kinetics and equilibrium studies.

    Science.gov (United States)

    Amosa, Mutiu K

    2016-08-01

    Sorption optimization and mechanism of hardness and alkalinity on bifunctional empty fruit bunch-based powdered activation carbon (PAC) were studied. The PAC possessed both high surface area and ion-exchange properties, and it was utilized in the treatment of biotreated palm oil mill effluent. Batch adsorption experiments designed with Design Expert(®) were conducted in correlating the singular and interactive effects of the three adsorption parameters: PAC dosage, agitation speed and contact time. The sorption trends of the two contaminants were sequentially assessed through a full factorial design with three factor interaction models and a central composite design with polynomial models of quadratic order. Analysis of variance revealed the significant factors on each design response with very high R(2) values indicating good agreement between model and experimental values. The optimum operating conditions of the two contaminants differed due to their different regions of operating interests, thus necessitating the utility of desirability factor to get consolidated optimum operation conditions. The equilibrium data for alkalinity and hardness sorption were better represented by the Langmuir isotherm, while the pseudo-second-order kinetic model described the adsorption rates and behavior better. It was concluded that chemisorption contributed majorly to the adsorption process.

  16. Comparative Study of the Preparation of Reducing Sugars Hydrolyzed from High-Lignin Lignocellulose Pretreated with Ionic Liquid, Alkaline Solution and Their Combination

    Directory of Open Access Journals (Sweden)

    Hanny F. Sangian

    2015-05-01

    Full Text Available The ionicliquid [MMIM][DMP] was synthesized from the reactants methyl imidazole [MIM] and trimethylphosphate [TMP] and verified using 1HNMR and FTIR. Coconut coir dust was pretreated with a 1% alkaline solution.Its crystalline structure increased significantly due to the dissolution of lignin and hemicelluloses under alkaline conditions, exposing the cellulose. After NaOH and IL were employed, the XRD showed that peak (002 decreased significantly and peak (101 almost vanished. This significant decrease in crystallinity was related to the alteration of the substrate from the cellulose I structure to the cellulose II structure. The pretreated substrates were hydrolyzed to convert them to reducing sugars by pure cellulase and xylanase,and the reaction was conducted at 60°C, pH 3, for 12 or 48 hours. The yields of sugar hydrolyzed from untreated and NaOH-pretreated substrates were 0.07 and 0.12 g sugar/g lignocellulose, respectively. Pretreatment with IL or the combination of NaOH+IL resulted in yields of reducing sugars of 0.11 and 0.13 g/g, respectively. These findings showed that IL pretreatment of the high-lignin lignocellulose is a new prospect for the economical manufacture of reducing sugars and bioethanol in the coming years.

  17. Protease inhibitor from Moringa oleifera with potential for use as therapeutic drug and as seafood preservative.

    Science.gov (United States)

    Bijina, B; Chellappan, Sreeja; Krishna, Jissa G; Basheer, Soorej M; Elyas, K K; Bahkali, Ali H; Chandrasekaran, M

    2011-07-01

    Protease inhibitors are well known to have several applications in medicine and biotechnology. Several plant sources are known to return potential protease inhibitors. In this study plants belonging to different families of Leguminosae, Malvaceae, Rutaceae, Graminae and Moringaceae were screened for the protease inhibitor. Among them Moringa oleifera, belonging to the family Moringaceae, recorded high level of protease inhibitor activity after ammonium sulfate fractionation. M. oleifera, which grows throughout most of the tropics and having several industrial and medicinal uses, was selected as a source of protease inhibitor since so far no reports were made on isolation of the protease inhibitor. Among the different parts of M. oleifera tested, the crude extract isolated from the mature leaves and seeds showed the highest level of inhibition against trypsin. Among the various extraction media evaluated, the crude extract prepared in phosphate buffer showed maximum recovery of the protease inhibitor. The protease inhibitor recorded high inhibitory activity toward the serine proteases thrombin, elastase, chymotrypsin and the cysteine proteases cathepsin B and papain which have more importance in pharmaceutical industry. The protease inhibitor also showed complete inhibition of activities of the commercially available proteases of Bacillus licheniformis and Aspergillus oryzae. However, inhibitory activities toward subtilisin, esperase, pronase E and proteinase K were negligible. Further, it was found that the protease inhibitor could prevent proteolysis in a commercially valuable shrimp Penaeus monodon during storage indicating the scope for its application as a seafood preservative. This is the first report on isolation of a protease inhibitor from M. oleifera.

  18. Combinatorial strategy of sorbitol feeding and low-temperature induction leads to high-level production of alkaline β-mannanase in Pichia pastoris.

    Science.gov (United States)

    Zhu, Taicheng; You, Lijin; Gong, Fuyu; Xie, Minfeng; Xue, Yanfen; Li, Yin; Ma, Yanhe

    2011-09-10

    A process for efficient production of an alkaline β-mannanases from Bacillus sp. N16-5 was established by heterologous expression using Pichia pastoris. A high producing strain was generated by removing the native β-mannanases signal peptide and increasing the copy number of the mature β-mannanases gene. High cell density fermentation of this strain in 1-L bioreactor led to a production level of 4164 U/mL after 96 h of induction. Sorbitol co-feeding and temperature-lowering strategies both increased the β-mannanase production levels. Combined usage of these two strategies achieved the most effective result-the enzyme level reached 6336 U/mL within 84 h, which to our best knowledge is the highest production level reported for the expression of extreme β-mannanase thus far. The strategy described in this work can also be adapted to express other important industrial enzymes with extreme properties.

  19. Production and characterization of a novel protease from Bacillus sp. RRM1 under solid state fermentation.

    Science.gov (United States)

    Rajkumar, Renganathan; Kothilmozhian, Jayappriyan; Ramasamy, Rengasamy

    2011-06-01

    A commercially important alkaline protease, produced by Bacillus sp. RRM1 isolated from the red seaweed Kappaphycus alvarezii (Doty) Doty ex Silva, was first recognized and characterized in the present study. Identification of the isolated bacterium was done using both biochemical characterization as well as 16S rRNA gene sequencing. The bacterial strain, Bacillus sp. RRM1, produced a high level of protease using easily available, inexpensive agricultural residues solid-state fermentation (SSF). Among them, wheat bran was found to be the best substrate. Influences of process parameters such as moistening agents, moisture level, temperature, inoculum concentration, and co-carbon and co-nitrogen sources on the fermentation were also evaluated. Under optimized conditions, maximum protease production (i.e., 2081 U/g) was obtained from wheat bran, which is about 2-fold greater than the initial conditions. The protease enzyme was stable over a temperature range of 30-60 degrees C and pH 6-12, with maximum activity at 50 degrees C and pH 9.0. Whereas the metal ions Na+, Ca2+, and K+ enhanced the activity of the enzyme, others such as Hg2+, Cu2+, Fe2+, Co2+, and Zn2+ had rendered negative effects. The activity of the enzyme was inhibited by EDTA and enhanced by Cu2+ ions, thus indicating the nature of the enzyme as a metalloprotease. The enzyme showed extreme stability and activity even in the presence of detergents, surfactants, and organic solvents. Moreover, the present findings opened new vistas in the utilization of wheat bran, a cheap, abundantly available, and effective waste as a substrate for SSF.

  20. Protease increases fermentation rate and ethanol yield in dry-grind ethanol production.

    Science.gov (United States)

    Johnston, David B; McAloon, Andrew J

    2014-02-01

    The effects of acid protease and urea addition during the fermentation step were evaluated. The fermentations were also tested with and without the addition of urea to determine if protease altered the nitrogen requirements of the yeast. Results show that the addition of the protease had a statistically significant effect on the fermentation rate and yield. Fermentation rates and yields were improved with the addition of the protease over the corresponding controls without protease. Protease addition either with or with added urea resulted in a higher final ethanol yield than without the protease addition. Urea addition levels >1200 ppm of supplemental nitrogen inhibited ethanol production. The economic effects of the protease addition were evaluated by using process engineering and economic models developed at the Eastern Regional Research Center. The decrease in overall processing costs from protease addition was as high as $0.01/L (4 ¢/gal) of denatured ethanol produced.

  1. Anodes for alkaline electrolysis

    Science.gov (United States)

    Soloveichik, Grigorii Lev

    2011-02-01

    A method of making an anode for alkaline electrolysis cells includes adsorption of precursor material on a carbonaceous material, conversion of the precursor material to hydroxide form and conversion of precursor material from hydroxide form to oxy-hydroxide form within the alkaline electrolysis cell.

  2. Alkaline detergent enzymes from alkaliphiles: enzymatic properties, genetics, and structures.

    Science.gov (United States)

    Ito, S; Kobayashi, T; Ara, K; Ozaki, K; Kawai, S; Hatada, Y

    1998-08-01

    The cleaning power of detergents seems to have peaked; all detergents contain similar ingredients and are based on similar detergency mechanisms. To improve detergency, modern types of heavy-duty powder detergents and automatic dishwasher detergents usually contain one or more enzymes, such as protease, amylase, cellulase, and lipase. Alkaliphilic Bacillus strains are often good sources of alkaline extracellular enzymes, the properties of which fulfil the essential requirements for enzymes to be used in detergents. We have isolated numbers of alkaliphilic Bacillus that produce such alkaline detergent enzymes, including cellulase (CMCase), protease, alpha-amylase, and debranching enzymes, and have succeeded in large-scale industrial production of some of these enzymes. Here, we describe the enzymatic properties, genetics, and structures of the detergent enzymes that we have developed.

  3. Survey of the rubber tree genome reveals a high number of cysteine protease-encoding genes homologous to Arabidopsis SAG12

    Science.gov (United States)

    Liu, Jianting; Yang, Lifu; Xie, Guishui

    2017-01-01

    Arabidopsis thaliana SAG12, a senescence-specific gene encoding a cysteine protease, is widely used as a molecular marker for the study of leaf senescence. To date, its potential orthologues have been isolated from several plant species such as Brassica napus and Nicotiana tabacum. However, little information is available in rubber tree (Hevea brasiliensis), a rubber-producing plant of the Euphorbiaceae family. This study presents the identification of SAG12-like genes from the rubber tree genome. Results showed that an unexpected high number of 17 rubber orthologues with a single intron were found, contrasting the single copy with two introns in Arabidopsis. The gene expansion was also observed in another two Euphorbiaceae plants, castor bean (Ricinus communis) and physic nut (Jatropha curcas), both of which contain 8 orthologues. In accordance with no occurrence of recent whole-genome duplication (WGD) events, most duplicates in castor and physic nut were resulted from tandem duplications. In contrast, the duplicated HbSAG12H genes were derived from tandem duplications as well as the recent WGD. Expression analysis showed that most HbSAG12H genes were lowly expressed in examined tissues except for root and male flower. Furthermore, HbSAG12H1 exhibits a strictly senescence-associated expression pattern in rubber tree leaves, and thus can be used as a marker gene for the study of senescence mechanism in Hevea. PMID:28166280

  4. Parallel ultra high pressure liquid chromatography-mass spectrometry for the quantification of HIV protease inhibitors using dried spot sample collection format.

    Science.gov (United States)

    Watanabe, Kyoko; Varesio, Emmanuel; Hopfgartner, Gérard

    2014-08-15

    An assay was developed and validated for the quantification of eight protease inhibitors (indinavir (IDV), ritonavir (RTV), lopinavir (LPV), saquinavir (SQV), amprenavir (APV), nelfinavir (NFV), atazanavir (AZV) and darunavir (DRV)) in dried plasma spots using parallel ultra-high performance liquid chromatography and mass spectrometry detection in the multiple reaction monitoring mode. For each analyte an isotopically labeled internal standard was used and the assay based on liquid-solid extraction the area response ratio (analyte/IS) was found to be linear; from 0.025 μg/ml to 20 μg/ml for IDV, SQV, DRV, AZV, LPV, from 0.025 μg/ml to 10 μg/ml for NFV, APV and from 0.025 μg/ml to 5 μg/ml for RTV using 15 μl of plasma spotted on filter paper placed in a sample tube. The total analysis time was of 4 min and inter-assay accuracies and precisions were in the range of 87.7-109% and 2.5-11.8%, respectively. On dried plasma spots all analytes were found to be stable for at least 7 days. Practicability of the assay to blood was also demonstrated. The sample drying process could be reduced to 5 min using a commercial microwave system without any analyte degradation. Together with quantification, confirmatory analysis was performed on representative clinical samples.

  5. Use of rosuvastatin to treat protease inhibitor-associated hypercholesterolaemia in a HIV-infected patient at high risk of cardiovascular diseases

    Directory of Open Access Journals (Sweden)

    Leonardo Calza

    2010-09-01

    Full Text Available Rosuvastatin represents one of the latest inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA reductase introduced in clinical practice for the treatment of hypercholesterolaemia. In comparative trials, across dose ranges this statin reduced low-density lipoprotein (LDL cholesterol and total cholesterol significantly more than atorvastatin, simvastatin, and pravastatin, and triglycerides significantly more than simvastatin and pravastatin. In healthy subjects with normal LDL cholesterol and elevated C-reactive protein, rosuvastatin treatment produced a significant decrease in the incidence of cardiovascular events.Its chemical and pharmacokinetic properties suggest a very limited penetration in extrahepatic tissues with a lower risk of muscle toxicity and metabolically mediated drug-drug interactions, suggesting a low risk of pharmacokinetic interactions with antiretroviral drugs in patients with HIV infection. We describe a case of protease inhibitor-associated hypercholesterolaemia in a male HIV-infected patient with high cardiovascular risk. Treatment with rosuvastatin leaded to a significant reduction in total and LDL cholesterol levels, with a good tolerability profile after 15 months of follow-up.

  6. Alkaline resistant ceramics; Alkalimotstaandskraftiga keramer

    Energy Technology Data Exchange (ETDEWEB)

    Westberg, Stig-Bjoern [Vattenfall Utveckling AB, Aelvkarleby (Sweden)

    2001-02-01

    Despite durability in several environments, ceramics and refractories can not endure alkaline environments at high temperature. An example of such an environment is when burning biofuel in modern heat and power plants in which the demand for increasing efficiency results in higher combustion temperatures and content of alkaline substances in the flue gas. Some experiences of these environments has been gained from such vastly different equipment as regenerator chambers in the glass industry and MHD-generators. The grains of a ceramic material are usually bonded together by a glassy phase which despite it frequently being a minor constituent render the materials properties and limits its use at elevated temperature. The damage is usually caused by alkaline containing low-melting phases and the decrease of the viscosity of the bonding glass phase which is caused by the alkaline. The surfaces which are exposed to the flue gas in a modern power plant are not only exposed to the high temperature but also a corroding and eroding, particle containing, gas flow of high velocity. The use of conventional refractory products is limited to 1300-1350 deg C. Higher strength and fracture toughness as well as durability against gases, slag and melts at temperatures exceeding 1700 deg C are expected of the materials of the future. Continuous transport of corrosive compounds to the surface and corrosion products from the surface as well as a suitable environment for the corrosion to occur in are prerequisites for extensive corrosion to come about. The highest corrosion rate is therefore found in a temperature interval between the dew point and the melting point of the alkaline-constituent containing compound. It is therefore important that the corrosion resistance is sufficient in the environment in which alkaline containing melts or slag may appear. In environments such as these, even under normal circumstances durable ceramics, such as alumina and silicon carbide, are attacked

  7. Proteases in Periodontal Disease

    Directory of Open Access Journals (Sweden)

    Ana Rita Sokolonski ANTON

    2006-09-01

    Full Text Available Introduction: The caries and the periodontal disease (PD are the most frequent alterations in the oral cavity. The PD presents two stages: gengivitis and periodontitis. The destruction of collagenous fibers which encases the tooth onto the alveolar bone is characteristic of the pariodontitis. The inclusion loss caused by this pathology is due to the presence of bacteria and their products, besides the tissue destruction. This process is caused by excessive discharge of cells of the organism defence which reach the damaged area, and among these cells are neutrophils. These cells free lysosomal granule, where enzymes known as proteases (elastase, colagenasis and catepsin G are present. When excessively delivered, they cause extensive tissue destruction. The organism innate defence respond to this process activating anti-proteases, such as alfa-1-antitripsin e alfa-2-macrogoblulin, and, as consequence, the inflammatory process is subdued. Objective: Revision of the literature on periodontitis and its markers. In periodontitis, the balance between protease and anti-protese seems to be altered and lead to the appearance of these ones. There is an increase of prevalence of PD in the world population. In recent times, it has been associated to systemic conditions that lead to tissue destruction. Perhaps, the cause is based on an exacerbated tissue reaction, more than on the bacterial aggression. Conclusion: The predisposition of the organism is an important factor for the disease development. At reading different studies, it was observed that the discharged protease during the neutrophils degranulation process has internal, not bacterial, origin.

  8. Death proteases come alive

    NARCIS (Netherlands)

    Woltering, E.J.

    2004-01-01

    Cell death in plants exhibits morphological features comparable to caspase-mediated apoptosis in animals, suggesting that plant cell death is executed by (caspase-like) proteases. However, to date, no caspase homologues have been identified in plants and therefore the existence and nature of these p

  9. Antarctic bacterial isolates that produce cold-active extracellular proteases at low temperature but are active and stable at high temperature

    Directory of Open Access Journals (Sweden)

    Cecilia Martínez-Rosales

    2011-04-01

    Full Text Available We report the isolation and identification of bacteria that produce extracellular cold-active proteases, obtained from water samples collected near the Uruguayan Antarctic Base on King George Island, South Shetlands. The bacteria belonged to the genera Pseudomonas (growth between 4 and 30 °C and Flavobacterium (growth between 4 and 18 °C. In all cases, extracellular protease production was evident when reaching the stationary phase at 18 and 4 °C but was not detected at 30 °C. The zymogram revealed the secretion of one extracellular protease per isolate, each with different relative electrophoretic mobility. The extracellular proteases produced at 4 °C showed thermal activity and stability at 30 °C. Both activity and stability at a temperature higher that 10 °C have no physiological meaning because the isolates do not experience such temperatures in the Antarctic environment; however, the possible ecological value of cold-active and -stable extracellular proteases is discussed.

  10. Petrogenesis and its significance to continental dynamics of the Neogene high-potassium calc-alkaline volcanic rock association from north Qiangtang, Tibetan Plateau

    Institute of Scientific and Technical Information of China (English)

    赖绍聪; 刘池阳; S.Y.O’Reilly

    2001-01-01

    Detailed studies indicate that the main rock type of the Neogene high-potassium calc-alkaline volcanic rock association from north Qiangtang is andesite, dacite and rhyolite. They belong to typical crust-generation magmatic system and originate from the special thickened crust of the Tibetan Plateau by dehydration melting. This group of rocks exhibits LREE enrichment but no remarkable Eu anomaly that shows their source region should be a thickened deep crust consisting of eclogitic mass group, implying that the crust had been thickened and an eclogitic deep crust had been formed during the Neogene period in Qiangtang area. This understanding is important and significant to making further discussion on the uplift mechanism and continental dynamics of the Tibetan Plateau.

  11. Cystatins--Extra- and intracellular cysteine protease inhibitors: High-level secretion and uptake of cystatin C in human neuroblastoma cells.

    Science.gov (United States)

    Wallin, Hanna; Bjarnadottir, Maria; Vogel, Lotte K; Wassélius, Johan; Ekström, Ulf; Abrahamson, Magnus

    2010-11-01

    Cystatins are present in mammals, birds, fish, insects, plants, fungi and protozoa and constitute a large protein family, with most members sharing a cysteine protease inhibitory function. In humans 12 functional cystatins exist, forming three groups based on molecular organisation and distribution in the organism. The type 1 cystatins (A and B) are known as intracellular, type 2 cystatins (C, D, E/M, F, G, S, SN and SA) extracellular and type 3 cystatins (L- and H-kininogen) intravascular proteins. The present paper is focused on the human cystatins and especially those of type 2, which are directed (with signal peptides) for cellular export following translation. Results indicating existence of systems for significant internalisation of type 2 cystatins from the extracellular to intracellular compartments are reviewed. Data showing that human neuroblastoma cell lines generally secrete high levels, but also contain high amounts of cystatin C are presented. Culturing of these cells in medium containing cystatin C at concentrations found in body fluids resulted in increased intracellular cystatin C, as a result of an uptake process. At immunofluorescence cytochemistry a pronounced vesicular cystatin C staining was observed. The simplistic denotation of the type 2 cystatins as extracellular inhibitors is thus challenged, and possible biological functions of the internalised cystatins are discussed. To illustrate the special case of high cellular cystatin content seen in cells of patients with hereditary cystatin C amyloid angiopathy, expression vectors for wild-type and L68Q mutated cystatin C were used to transfect SK-N-BE(2) cells. Clones overexpressing the two variants showed increased secreted levels of cystatin C. Within the cells the L68Q variant appeared to mainly localise to the endoplasmic reticulum rather than to acidic vesicular organelles, indicating limitations in the transport out from the cell rather than increased uptake as explanation for the

  12. Alkaline battery operational methodology

    Science.gov (United States)

    Sholklapper, Tal; Gallaway, Joshua; Steingart, Daniel; Ingale, Nilesh; Nyce, Michael

    2016-08-16

    Methods of using specific operational charge and discharge parameters to extend the life of alkaline batteries are disclosed. The methods can be used with any commercial primary or secondary alkaline battery, as well as with newer alkaline battery designs, including batteries with flowing electrolyte. The methods include cycling batteries within a narrow operating voltage window, with minimum and maximum cut-off voltages that are set based on battery characteristics and environmental conditions. The narrow voltage window decreases available capacity but allows the batteries to be cycled for hundreds or thousands of times.

  13. High Pressure Homogenization Of Porcine Pepsin Protease: Effects On Enzyme Activity, Stability, Milk Coagulation Profile And Gel Development.

    OpenAIRE

    Bruno Ricardo de Castro Leite Júnior; Alline Artigiani Lima Tribst; Marcelo Cristianini

    2015-01-01

    This study investigated the effect of high pressure homogenization (HPH) (up to 190 MPa) on porcine pepsin (proteolytic and milk-clotting activities), and the consequences of using the processed enzyme in milk coagulation and gel formation (rheological profile, proteolysis, syneresis, and microstructure). Although the proteolytic activity (PA) was not altered immediately after the HPH process, it reduced during enzyme storage, with a 5% decrease after 60 days of storage for samples obtained w...

  14. A novel protease activity assay using a protease-responsive chaperone protein

    Energy Technology Data Exchange (ETDEWEB)

    Sao, Kentaro [Graduate School of Systems Life Sciences, Kyushu University, 744 Motooka Nishi-ku, Fukuoka 819-0395 (Japan); Murata, Masaharu, E-mail: m-murata@dem.med.kyushu-u.ac.jp [Department of Advanced Medical Initiatives, Faculty of Medical Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku Fukuoka 812-8582 (Japan); Fujisaki, Yuri; Umezaki, Kaori [Department of Advanced Medical Initiatives, Faculty of Medical Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku Fukuoka 812-8582 (Japan); Mori, Takeshi; Niidome, Takuro; Katayama, Yoshiki [Graduate School of Systems Life Sciences, Kyushu University, 744 Motooka Nishi-ku, Fukuoka 819-0395 (Japan); Department of Applied Chemistry, Faculty of Engineering, Kyushu University, Nishi-ku Fukuoka 819-0395 (Japan); Center for Future Chemistry, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395 (Japan); Hashizume, Makoto [Department of Advanced Medical Initiatives, Faculty of Medical Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku Fukuoka 812-8582 (Japan)

    2009-06-05

    Protease activity assays are important for elucidating protease function and for developing new therapeutic agents. In this study, a novel turbidimetric method for determining the protease activity using a protease-responsive chaperone protein is described. For this purpose, a recombinant small heat-shock protein (sHSP) with an introduced Factor Xa protease recognition site was synthesized in bacteria. This recombinant mutant, FXa-HSP, exhibited chaperone-like activity at high temperatures in cell lysates. However, the chaperone-like activity of FXa-HSP decreased dramatically following treatment with Factor Xa. Protein precipitation was subsequently observed in the cell lysates. The reaction was Factor Xa concentration-dependent and was quantitatively suppressed by a specific inhibitor for Factor Xa. Protein aggregation was detected by a simple method based on turbidimetry. The results clearly demonstrate that this assay is an effective, easy-to-use method for determining protease activities without the requirement of labeling procedures and the use of radioisotopes.

  15. Nutritional value and proteases of Lentinus citrinus produced by solid state fermentation of lignocellulosic waste from tropical region

    Directory of Open Access Journals (Sweden)

    Ana Rita Gaia Machado

    2016-09-01

    Full Text Available This paper examined the growth and yield performance of Lentinus citrinus on cupuaçu exocarp (Theobroma grandiflorum mixed with litter (CE + LI or rice bran (Oryza sativa (CE + RB in the ratio of 2:1 (800 g:200 g to investigate the nutritional composition and proteolytic potential of the fruiting body produced. Significance values of yield were determined on substrate combinations. In CE + LI the biological efficiency of the mushrooms was 93.5% and the content of fat (4.5%, fiber (11.0%, protein (27.0% and amino acids were higher when compared with CE + RB. Among the amino acids, the amount of glutamic acid, aspartic acid, alanine, arginine and leucine was high. The biological efficiency on CE + RB reduced to 84.2% and based on the nutritional value, carbohydrates (53.59%, energy (324.33 kcal and minerals such as zinc, iron, copper, potassium and phosphorus were higher in this substrate combination. Protease activity from fruiting body was significant in CE + LI (463.55 U/mL. This protease showed an optimal activity at 50 °C in neutral and alkaline pH with maximum stability at 30 °C at alkaline pH. This is the first report of L. citrinus fruiting body nutritional composition with potential for human food and application in industrial processes.

  16. Nutritional value and proteases of Lentinus citrinus produced by solid state fermentation of lignocellulosic waste from tropical region.

    Science.gov (United States)

    Machado, Ana Rita Gaia; Teixeira, Maria Francisca Simas; de Souza Kirsch, Larissa; Campelo, Maria da Conceição Loureiro; de Aguiar Oliveira, Ila Maria

    2016-09-01

    This paper examined the growth and yield performance of Lentinus citrinus on cupuaçu exocarp (Theobroma grandiflorum) mixed with litter (CE + LI) or rice bran (Oryza sativa) (CE + RB) in the ratio of 2:1 (800 g:200 g) to investigate the nutritional composition and proteolytic potential of the fruiting body produced. Significance values of yield were determined on substrate combinations. In CE + LI the biological efficiency of the mushrooms was 93.5% and the content of fat (4.5%), fiber (11.0%), protein (27.0%) and amino acids were higher when compared with CE + RB. Among the amino acids, the amount of glutamic acid, aspartic acid, alanine, arginine and leucine was high. The biological efficiency on CE + RB reduced to 84.2% and based on the nutritional value, carbohydrates (53.59%), energy (324.33 kcal) and minerals such as zinc, iron, copper, potassium and phosphorus were higher in this substrate combination. Protease activity from fruiting body was significant in CE + LI (463.55 U/mL). This protease showed an optimal activity at 50 °C in neutral and alkaline pH with maximum stability at 30 °C at alkaline pH. This is the first report of L. citrinus fruiting body nutritional composition with potential for human food and application in industrial processes.

  17. Uranium in alkaline rocks

    Energy Technology Data Exchange (ETDEWEB)

    Murphy, M.; Wollenberg, H.; Strisower, B.; Bowman, H.; Flexser, S.; Carmichael, I.

    1978-04-01

    Geologic and geochemical criteria were developed for the occurrence of economic uranium deposits in alkaline igneous rocks. A literature search, a limited chemical analytical program, and visits to three prominent alkaline-rock localities (Ilimaussaq, Greenland; Pocos de Caldas, Brazil; and Powderhorn, Colorado) were made to establish criteria to determine if a site had some uranium resource potential. From the literature, four alkaline-intrusive occurrences of differing character were identified as type-localities for uranium mineralization, and the important aspects of these localities were described. These characteristics were used to categorize and evaluate U.S. occurrences. The literature search disclosed 69 U.S. sites, encompassing nepheline syenite, alkaline granite, and carbonatite. It was possible to compare two-thirds of these sites to the type localities. A ranking system identified ten of the sites as most likely to have uranium resource potential.

  18. A study of photosynthetic biogas upgrading based on a high rate algal pond under alkaline conditions: Influence of the illumination regime.

    Science.gov (United States)

    Franco-Morgado, Mariana; Alcántara, Cynthia; Noyola, Adalberto; Muñoz, Raúl; González-Sánchez, Armando

    2017-08-15

    Microalgal-bacterial processes have emerged as environmental friendly systems for the cost-effective treatment of anaerobic effluents such as biogas and nutrients-laden digestates. Environmental parameters such as temperature, irradiation, nutrient concentration and pH effect the performance of the systems. In this paper, the potential of a microalgal-bacterial photobioreactor operated under high pH (≈9.5) and high alkalinity to convert biogas into biomethane was evaluated. The influence of the illumination regime (continuous light supply vs 12h/12h light/dark cycles) on the synthetic biogas upgrading efficiency, biomass productivity and nutrient removal efficiency was assessed in a High-Rate Algal Pond interconnected to a biogas absorption bubble column. No significant differences in the removal efficiency of CO2 and H2S (91.5±2% and 99.5%±0.5, respectively) were recorded regardless of the illumination regime. The high fluctuations of the dissolved oxygen concentration during operation under light/dark cycles allowed to evaluate the specific growth rate and the specific partial degradation rate of the microalgae biomass by photosynthesis and respiration, respectively. The respiration reduced the net microalgae biomass productivity under light/dark cycles compared with process operation under the continuous light supply. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. 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.

  20. High Pressure Homogenization of Porcine Pepsin Protease: Effects on Enzyme Activity, Stability, Milk Coagulation Profile and Gel Development.

    Directory of Open Access Journals (Sweden)

    Bruno Ricardo de Castro Leite Júnior

    Full Text Available This study investigated the effect of high pressure homogenization (HPH (up to 190 MPa on porcine pepsin (proteolytic and milk-clotting activities, and the consequences of using the processed enzyme in milk coagulation and gel formation (rheological profile, proteolysis, syneresis, and microstructure. Although the proteolytic activity (PA was not altered immediately after the HPH process, it reduced during enzyme storage, with a 5% decrease after 60 days of storage for samples obtained with the enzyme processed at 50, 100 and 150 MPa. HPH increased the milk-clotting activity (MCA of the enzyme processed at 150 MPa, being 15% higher than the MCA of non-processed samples after 60 days of storage. The enzyme processed at 150 MPa produced faster aggregation and a more consistent milk gel (G' value 92% higher after 90 minutes when compared with the non-processed enzyme. In addition, the gels produced with the enzyme processed at 150 MPa showed greater syneresis after 40 minutes of coagulation (forming a more compact protein network and lower porosity (evidenced by confocal microscopy. These effects on the milk gel can be associated with the increment in MCA and reduction in PA caused by the effects of HPH on pepsin during storage. According to the results, HPH stands out as a process capable of changing the proteolytic characteristics of porcine pepsin, with improvements on the milk coagulation step and gel characteristics. Therefore, the porcine pepsin submitted to HPH process can be a suitable alternative for the production of cheese.

  1. High Pressure Homogenization of Porcine Pepsin Protease: Effects on Enzyme Activity, Stability, Milk Coagulation Profile and Gel Development.

    Science.gov (United States)

    Leite Júnior, Bruno Ricardo de Castro; Tribst, Alline Artigiani Lima; Cristianini, Marcelo

    2015-01-01

    This study investigated the effect of high pressure homogenization (HPH) (up to 190 MPa) on porcine pepsin (proteolytic and milk-clotting activities), and the consequences of using the processed enzyme in milk coagulation and gel formation (rheological profile, proteolysis, syneresis, and microstructure). Although the proteolytic activity (PA) was not altered immediately after the HPH process, it reduced during enzyme storage, with a 5% decrease after 60 days of storage for samples obtained with the enzyme processed at 50, 100 and 150 MPa. HPH increased the milk-clotting activity (MCA) of the enzyme processed at 150 MPa, being 15% higher than the MCA of non-processed samples after 60 days of storage. The enzyme processed at 150 MPa produced faster aggregation and a more consistent milk gel (G' value 92% higher after 90 minutes) when compared with the non-processed enzyme. In addition, the gels produced with the enzyme processed at 150 MPa showed greater syneresis after 40 minutes of coagulation (forming a more compact protein network) and lower porosity (evidenced by confocal microscopy). These effects on the milk gel can be associated with the increment in MCA and reduction in PA caused by the effects of HPH on pepsin during storage. According to the results, HPH stands out as a process capable of changing the proteolytic characteristics of porcine pepsin, with improvements on the milk coagulation step and gel characteristics. Therefore, the porcine pepsin submitted to HPH process can be a suitable alternative for the production of cheese.

  2. From proteases to proteomics.

    Science.gov (United States)

    Neurath, H

    2001-04-01

    This personal and professional autobiography covers the 50-yr period of 1950-2000 and includes the following topics: History of the University of Washington School of Medicine and its Department of Biochemistry (Mount Rainier and the University of Washington, recruiting faculty, biology, research programs); scientific editing (publication, Biochemistry, Protein Science, electronic publication); Europe revisited (Heidelberg, approaching retirement, the German Research Center, reunion in Vienna); and 50 yr of research on proteolytic enzymes (trypsin, carboxypeptidases, mast cell proteases, future developments).

  3. Correlation Between Expression of High Temperature Requirement Serine Protease A1 (HtrA1) in Nucleus Pulposus and T2 Value of Magnetic Resonance Imaging.

    Science.gov (United States)

    Li, Dapeng; Yue, Jiawei; Jiang, Lu; Huang, Yonghui; Sun, Jifu; Wu, Yan

    2017-04-22

    BACKGROUND Degrading enzymes play an important role in the process of disc degeneration. The objective of this study was to investigate the correlation between the expression of high temperature requirement serine protease A1 (HtrA1) in the nucleus pulposus and the T2 value of the nucleus pulposus region in magnetic resonance imaging (MRI). MATERIAL AND METHODS Thirty-six patients who had undergone surgical excision of the nucleus pulposus were examined by MRI before surgery. Pfirrmann grading of the target intervertebral disc was performed according to the sagittal T2-weighted imaging, and the T2 value of the target nucleus pulposus was measured according to the median sagittal T2 mapping. The correlation between the Pfirrmann grade and the T2 value was analyzed. The expression of HtrA1 in the nucleus pulposus was analyzed by RT-PCR and Western blot. The correlation between the expression of HtrA1 and the T2 value was analyzed. RESULTS The T2 value of the nucleus pulposus region was 33.11-167.91 ms, with an average of 86.64±38.73 ms. According to Spearman correlation analysis, there was a rank correlation between T2 value and Pfirrmann grade (Pcorrelation coefficient (rs)=-0.93617. There was a linear correlation between the mRNA level of HtrA1 and T2 value in nucleus pulposus tissues (a=3.88, b=-0.019, F=112.63, Pcorrelation between the expression level of HtrA1 protein and the T2 value in the nucleus pulposus tissues (a=3.30, b=-0.016, F=93.15, P<0.0001) and normalized regression coefficient=-0.86. CONCLUSIONS The expression of HtrA1 was strongly related to the T2 value, suggesting that HtrA1 plays an important role in the pathological process of intervertebral disc degeneration.

  4. A novel carboxyl-terminal protease derived from Paenibacillus lautus CHN26 exhibiting high activities at multiple sites of substrates

    DEFF Research Database (Denmark)

    Li, Yunxia; Pan, Yingjie; She, Qunxin;

    2013-01-01

    Carboxyl-terminal protease (CtpA) plays essential functions in posttranslational protein processing in prokaryotic and eukaryotic cells. To date, only a few bacterial ctpA genes have been characterized. Here we cloned and characterized a novel CtpA. The encoding gene, ctpAp (ctpA of Paenibacillus...

  5. Involvement of CD36 and intestinal alkaline phosphatases in fatty acid transport in enterocytes, and the response to a high-fat diet.

    Science.gov (United States)

    Lynes, Matthew D; Widmaier, Eric P

    2011-02-28

    The vertebrate intestine is notable for its plasticity in response to environmental, pathologic, reproductive, and dietary challenges. The molecular mechanisms of intestinal adaptations typically involve both morphologic and functional changes. In response to chronic ingestion of a high-fat diet, for example, the mammalian small intestine quickly adapts to efficiently accommodate increased transport of long-chain fatty acids across the mucosa. Whereas this may be adaptive in the short term, in the long term it may contribute to the pathologies associated with chronic high-fat diets in humans and other mammals. This review focuses on some of the known and putative mechanisms by which fatty acids are transported across the intestinal epithelium in addition to simple diffusion, and how these mechanisms may be regulated in part by a high-fat diet. A model is proposed in which two key proteins, CD36 and the enzyme intestinal alkaline phosphatase, work in a coordinated manner to optimize fatty acid transport across enterocytes in mice. Copyright © 2010 Elsevier Inc. All rights reserved.

  6. High serum alkaline phosphatase cooperating with MMP-9 predicts metastasis and poor prognosis in patients with primary osteosarcoma in Southern China

    Directory of Open Access Journals (Sweden)

    Han Ju

    2012-02-01

    Full Text Available Abstract Background Osteosarcoma is a malignant tumor with high ability to form invasion and metastasis. Identifying prognostic factor in osteosarcoma is helpful to select those patients for more aggressive management. Our study evaluated serum alkaline phosphatase (ALP cooperating with matrix metalloproteinase-9 (MMP-9 as an important prognostic predictor for local recurrence and distant metastasis of osteosarcoma. Methods 177 cases were included from the osteosarcoma patients treated at 1st Affiliated Hospital of Sun Yat-sen University (1999-2008. Pre-chemotherapy serum ALP (pre-ALP were studied and correlated with tumor recurrence, lung metastasis and patient survival. MMP-9 protein in tumor tissues was detected by immunohistochemistry and correlated with pre-ALP level. Results Pre-ALP were partitioned into normal, high, and very high groups, in each group the incidence of metastases was 12.2%, 21.2% and 34.6%, respectively (p = 0.007. In the three groups the mean disease-free survival (DFS was 57 ± 3.15, 28 ± 3.57 and 14 ± 3.35 months, respectively (p Conclusions Pre-ALP was an independent prognostic factor for the survival of osteosarcoma patients in south China, and correlated with MMP-9 expression and lung metastasis. ALP can also serve as a prognostic marker for treatment, and merit large-scale validation studies.

  7. High serum alkaline phosphatase cooperating with MMP-9 predicts metastasis and poor prognosis in patients with primary osteosarcoma in Southern China.

    Science.gov (United States)

    Han, Ju; Yong, Bicheng; Luo, Canqiao; Tan, Pingxian; Peng, Tingsheng; Shen, Jingnan

    2012-02-15

    Osteosarcoma is a malignant tumor with high ability to form invasion and metastasis. Identifying prognostic factor in osteosarcoma is helpful to select those patients for more aggressive management. Our study evaluated serum alkaline phosphatase (ALP) cooperating with matrix metalloproteinase-9 (MMP-9) as an important prognostic predictor for local recurrence and distant metastasis of osteosarcoma. 177 cases were included from the osteosarcoma patients treated at 1st Affiliated Hospital of Sun Yat-sen University (1999-2008). Pre-chemotherapy serum ALP (pre-ALP) were studied and correlated with tumor recurrence, lung metastasis and patient survival. MMP-9 protein in tumor tissues was detected by immunohistochemistry and correlated with pre-ALP level. Pre-ALP were partitioned into normal, high, and very high groups, in each group the incidence of metastases was 12.2%, 21.2% and 34.6%, respectively (p = 0.007). In the three groups the mean disease-free survival (DFS) was 57 ± 3.15, 28 ± 3.57 and 14 ± 3.35 months, respectively (p lung metastasis rate decreased (p = 0.028); DFS and OS were both prolonged (p osteosarcoma patients in south China, and correlated with MMP-9 expression and lung metastasis. ALP can also serve as a prognostic marker for treatment, and merit large-scale validation studies.

  8. Association of alkaline phosphatase phenotypes with arthritides

    Directory of Open Access Journals (Sweden)

    Padmini A

    2004-01-01

    Full Text Available Arthritides, a symmetrical polyarticular disease of the bone are a heterogenous group of disorders in which hereditary and environmental factors in combination with an altered immune response appear to play a causative and pathogenic role in its occurrence. Alkaline phosphatase (ALP is an enzyme found in all tissues, with particularly high concentrations of ALP observed in the liver, bile ducts, placenta, and bone.Alkaline phosphatase is an orthophosphoric monoester phosphohydrolase catalyzing the hydrolysis of organic esters at alkaline pH, indicating that alkaline phosphatase is involved in fundamental biological processes.1 The present study envisages on identifying the specific electromorphic association of alkaline phosphatase with arthritides. Phenotyping of serum samples was carried out by PAGE (Polyacrylamide gel electrophoresis following Davies (19642 protocol on 41 juvenile arthritis, 150 rheumatoid arthritis and 100 osteo arthritis apart from, 25 normal children and 100 adult healthy subjects. Phenotyping of alkaline phosphatase revealed an increase in preponderance of p+ and p++ phenotypes in juvenile, rheumatoid and osteo arthritic patients. However a significant association of these phenotypes was observed only with rheumatoid arthritis condition (c2:17.46. Similarly, a significant increase of p+ phenotypes in female rheumatoid arthritis patients was observed (c2:14.973, suggesting that the decrease in p° (tissue non specific synthesis/secretion of alkaline phosphatase could be associated with decreased mineralization and ossification process in arthritis condition.

  9. Development of alkaline fuel cells.

    Energy Technology Data Exchange (ETDEWEB)

    Hibbs, Michael R.; Jenkins, Janelle E.; Alam, Todd Michael; Janarthanan, Rajeswari; Horan, James L.; Caire, Benjamin R.; Ziegler, Zachary C.; Herring, Andrew M.; Yang, Yuan; Zuo, Xiaobing; Robson, Michael H.; Artyushkova, Kateryna; Patterson, Wendy; Atanassov, Plamen Borissov

    2013-09-01

    This project focuses on the development and demonstration of anion exchange membrane (AEM) fuel cells for portable power applications. Novel polymeric anion exchange membranes and ionomers with high chemical stabilities were prepared characterized by researchers at Sandia National Laboratories. Durable, non-precious metal catalysts were prepared by Dr. Plamen Atanassovs research group at the University of New Mexico by utilizing an aerosol-based process to prepare templated nano-structures. Dr. Andy Herrings group at the Colorado School of Mines combined all of these materials to fabricate and test membrane electrode assemblies for single cell testing in a methanol-fueled alkaline system. The highest power density achieved in this study was 54 mW/cm2 which was 90% of the project target and the highest reported power density for a direct methanol alkaline fuel cell.

  10. 毛霉高产蛋白酶菌株产酶条件的优化%Optimization of the Producing Conditions of Enzyme for Mucor With High Productive Protease Strain

    Institute of Scientific and Technical Information of China (English)

    刘芳; 曹新志; 游见明; 刘春明

    2012-01-01

    The article studied the producing conditions of enzyme for Mucor with high productive protease strain that was induced mutation by UV. The purpose was to improve enzyme activity further. The results of single factor experiments and orthogonal tests indicated that the optimal culture medium initial pH value, proportion of bran and soybean meal, ratio of material and water, culture temperature, culture time for this excellent mutant strain to produce protease were 7.0, 7.3,1:1.1, 28℃ 3d. The strain's protease activity was 180.529U/g after optimization, which had increased by 54%.%对经紫外线诱变选育的毛霉高产蛋白酶菌株进行产酶条件优化,使酶活得到进一步提高.单因素试验和正交试验得该优良突变菌株的最优产酶条件为:培养基初始pH 7.0,麸皮豆粕比7∶3,料水比1∶1.1,培养温度28℃,培养时间3d.优化后蛋白酶活力达180.529U/g,比优化前提高了54%.

  11. Nucleic Acid Aptamers Against Proteases

    DEFF Research Database (Denmark)

    Dupont, D M; Andersen, L M; Bøtkjær, Kenneth Alrø

    2011-01-01

    Proteases are potential or realized therapeutic targets in a wide variety of pathological conditions. Moreover, proteases are classical subjects for studies of enzymatic and regulatory mechanisms. We here review the literature on nucleic acid aptamers selected with proteases as targets. Designing...... strategies and of new principles for regulating the activity of the inhibitory action of aptamers of general interest to researchers working with nucleic acid aptamers...

  12. Cathepsin proteases in Toxoplasma gondii

    OpenAIRE

    Dou, Zhicheng; Carruthers, Vern B.

    2011-01-01

    Cysteine proteases are important for the growth and survival of apicomplexan parasites that infect humans. The apicomplexan Toxoplasma gondii expresses five members of the C1 family of cysteine proteases, including one cathepsin L-like (TgCPL), one cathepsin B-like (TgCPB), and three cathepsin C-like (TgCPC1, 2 and 3) proteases. Recent genetic, biochemical and structural studies reveal that cathepsins function in microneme and rhoptry protein maturation, host cell invasion, replication, and n...

  13. Bottom-up synthesis of high-performance nitrogen-enriched transition metal/graphene oxygen reduction electrocatalysts both in alkaline and acidic solution

    Science.gov (United States)

    Lai, Qingxue; Gao, Qingwen; Su, Qi; Liang, Yanyu; Wang, Yuxi; Yang, Zhi

    2015-08-01

    Oxygen reduction electrocatalysts with low cost and excellent performance are urgently required for large-scale application in fuel cells and metal-air batteries. Though nitrogen-enriched transition metal/graphene hybrids (N-TM/G, TM = Fe, Co, and Ni and related compounds) have been developed as novel substitutes for precious metal catalysts (PMCs) towards oxygen reduction reaction (ORR), a significant challenge still remains for simple and efficient synthesis of N-TM/G catalysts with satisfactory electrocatalytic behavior. Herein, we demonstrate a universal bottom-up strategy for efficient fabrication of strongly-coupled N-TM/G catalysts. This strategy is implemented via direct polymerization of transition metal phthalocyanine (TMPc) in the two-dimensional confined space of in situ generated g-C3N4 and a subsequent pyrolysis. Such a space-confined bottom-up synthesis route successfully constructs a strongly-coupled triple junction of transition metal-graphitic carbon-nitrogen-doped graphene (TM-GC-NG) with extensive controllability over the specific surface area, nitrogen content/types as well as the states of metal. As a result, the optimized N-Fe/G materials have promising potential as high-performance NPMCs towards ORR both in alkaline and acidic solution.Oxygen reduction electrocatalysts with low cost and excellent performance are urgently required for large-scale application in fuel cells and metal-air batteries. Though nitrogen-enriched transition metal/graphene hybrids (N-TM/G, TM = Fe, Co, and Ni and related compounds) have been developed as novel substitutes for precious metal catalysts (PMCs) towards oxygen reduction reaction (ORR), a significant challenge still remains for simple and efficient synthesis of N-TM/G catalysts with satisfactory electrocatalytic behavior. Herein, we demonstrate a universal bottom-up strategy for efficient fabrication of strongly-coupled N-TM/G catalysts. This strategy is implemented via direct polymerization of transition

  14. Second generation of pseudotype-based serum neutralization assay for Nipah virus antibodies: sensitive and high-throughput analysis utilizing secreted alkaline phosphatase.

    Science.gov (United States)

    Kaku, Yoshihiro; Noguchi, Akira; Marsh, Glenn A; Barr, Jennifer A; Okutani, Akiko; Hotta, Kozue; Bazartseren, Boldbaatar; Fukushi, Shuetsu; Broder, Christopher C; Yamada, Akio; Inoue, Satoshi; Wang, Lin-Fa

    2012-01-01

    Nipah virus (NiV), Paramyxoviridae, Henipavirus, is classified as a biosafety level (BSL) 4 pathogen, along with the closely related Hendra virus (HeV). A novel serum neutralization test was developed for measuring NiV neutralizing antibodies under BSL2 conditions using a recombinant vesicular stomatitis virus (VSV) expressing secreted alkaline phosphatase (SEAP) and pseudotyped with NiV F/G proteins (VSV-NiV-SEAP). A unique characteristic of this novel assay is the ability to obtain neutralization titers by measuring SEAP activity in supernatant using a common ELISA plate reader. This confers a remarkable advantage over the first generation of NiV-pseudotypes expressing green fluorescent protein or luciferase, which require expensive and specific measuring equipment. Using panels of NiV- and HeV-specific sera from various species, the VSV-NiV-SEAP assay demonstrated neutralizing antibody status (positive/negative) consistent with that obtained by conventional live NiV test, and gave higher antibody titers than the latter. Additionally, when screening sixty-six fruit bat sera at one dilution, the VSV-NiV-SEAP assay produced identical results to the live NiV test and only required a very small amount (2μl) of sera. The results suggest that this novel VSV-NiV-SEAP assay is safe, useful for high-throughput screening of sera using an ELISA plate reader, and has high sensitivity and specificity.

  15. Nucleic Acid Aptamers Against Proteases

    DEFF Research Database (Denmark)

    Dupont, D M; Andersen, L M; Bøtkjær, Kenneth Alrø

    2011-01-01

    -specifically, for instance with vastly different affinities to zymogen and active enzyme forms. Furthermore, aptamers can be selected to inhibit the enzyme activity of the target proteases, but also to inhibit functionally important exosite interactions, for instance cofactor binding. Several protease-inhibiting aptamers......, directed against blood coagulation factors, are in clinical trials as anticoagulant drugs. Several of the studies on protease-binding aptamers have been pioneering and trend-setting in the field. The work with protease-binding aptamers also demonstrates many interesting examples of non-standard selection...

  16. Optimal conditions for production of extracellular protease from newly isolated Bacillus cereus strain CA15

    Directory of Open Access Journals (Sweden)

    Fikret Uyar

    2011-02-01

    Full Text Available An alkaline protease producer Bacillus sp. strain CA15 was isolated from soil. The microorganism was found to be closely related to Bacillus cereus based on 16S ribosomal DNA sequencing. The culture conditions for higher protease production were optimized with respect to carbon and nitrogen sources, metal ions, pH and temperature. Maximum protease production was obtained in the medium supplemented with 1% skim milk, 1% starch and 0.6% MgSO4.7H2O, initial pH 8.0 at 35oC. The best enzyme production was obtained during the stationary phase in which the cell density reached to 1.8x108 cells/mL. The level of protease was found to be low in the presence of inorganic nitrogen sources. The protease production was diminished in the presence of sucrose and lactose. The extreme stability towards Triton X-100, Tween 20 and SDS was observed by Bacillus sp. CA15 alkaline protease. The enzyme activity was inhibited by PMSF suggested that presence of serine residues at the active sites.

  17. Genome and secretome analyses provide insights into keratin decomposition by novel proteases from the non-pathogenic fungus Onygena corvina

    DEFF Research Database (Denmark)

    Huang, Yuhong; Kamp Busk, Peter; Herbst, Florian Alexander

    2015-01-01

    of proteases in keratin-degrading and non-keratin-degrading fungi indicated that 18 putative secreted proteases from four protease families (M36, M35, M43, and S8) may be responsible for keratin decomposition. Twelve of the 18 predicted protease genes could be amplified from O. corvina grown on keratinaceous...... materials and were transformed into Pichia pastoris. One of the recombinant proteases belonging to the S8 family showed high keratin-degrading activity. Furthermore, 29 different proteases were identified by mass spectrometry in the culture broth of O. corvina grown on feathers and bristle. The culture...... broth was fractionated by ion exchange chromatography to isolate active fractions with five novel proteases belonging to three protease families (S8, M28, and M3). Enzyme blends composed of three of these five proteases, one from each family, showed high degree of degradation of keratin in vitro...

  18. High levels of both serum gamma-glutamyl transferase and alkaline phosphatase are independent preictors of mortality in patients with stage 4-5 chronic kidney disease.

    Science.gov (United States)

    Caravaca-Fontán, Fernando; Azevedo, Lilia; Bayo, Miguel Ángel; Gonzales-Candia, Boris; Luna, Enrique; Caravaca, Francisco

    High serum gamma-glutamyl transferase (GGT) levels are associated with increased mortality in the general population. However, this association has scarcely been investigated in patients with chronic kidney disease (CKD). This study aims to investigate the clinical characteristics of CKD patients with abnormally elevated serum GGT, and its value for predicting mortality. Retrospective observational study in a population cohort of adults with stage 4-5 CKD not yet on dialysis. Demographic, clinical, and biochemical parameters of prognostic interest were recorded and used to characterise CKD patients with high levels of GGT (>36 IU/l). Cox proportional hazard regression models were used to analyse the influence of baseline serum GGT and alkaline phosphatase (ALP) levels on mortality for whatever reason. The study group consisted of 909 patients (mean age 65±15 years). Abnormally elevated GGT or ALP levels at baseline were observed in 209 (23%) and 172 (19%) patients, respectively, and concomitant elevations of GGT and ALP in 68 (7%). High GGT levels were associated with higher comorbidity burden, and a biochemical profile characterised by higher serum concentration of uric acid, triglycerides, alanine aminotransferase, ferritin, and C-reactive. During the study period, 365 patients (40%) died (median survival time=74 months). In adjusted Cox regression models, high levels of GGT (hazard ratio [HR]=1.39;CI 95%: 1.09-1.78, P=.009) and ALP (HR=1.31; CI95%: 1.02-1.68, P=.038) were independently associated with mortality. High serum levels of GGT are independent predictors of mortality in CKD patients. Copyright © 2016 Sociedad Española de Nefrología. Published by Elsevier España, S.L.U. All rights reserved.

  19. High background radiation areas of Ram sar in Iran: evaluation of DNA damage by alkaline single cell gel electrophoresis (SCE)

    Energy Technology Data Exchange (ETDEWEB)

    Masoomi, J.R. [Biophysics Department, College of Sciences, Tarbiat Modarres University, Tehran (Iran, Islamic Republic of); Mohammadi, Sh. [Radiation Molecular Genetic Laboratory, National Radiation Protection Department (NRPD), Iranian Nuclear Regulatory Authority (INRA), P.O. Box 14155-4494, Tehran (Iran, Islamic Republic of); Amini, M. [Faculty of Pharmacy, Azad University of Tehran (Iran, Islamic Republic of); Ghiassi-Nejad, M. [Biophysics Department, College of Sciences, Tarbiat Modarres University, Tehran (Iran, Islamic Republic of)]. E-mail: ghiassi@mailcity.com

    2006-07-01

    The hot springs in special areas in Ram sar, a northern coastal town in Iran, contain {sup 226}Ra and {sup 222}Rn. The natural radiation effects, radiosensitivity or adaptive responses, on the inhabitants of high natural radiation in Ram sar were studied. The single cell gel electrophoresis was used to monitor DNA damages. Three groups of volunteers were selected, one from high natural background radiation areas as the case group and two from normal background radiation areas as controls (control 1 and control 2). The latter one had the similar living situation to case group while the other (control 2) had different living situation from the other groups. Peripheral blood mononuclear cells (PMNCs) were separated and irradiated by {sup 6}Co source at five different gamma doses. It was found that the spontaneous level of DNA damage and the induced DNA damage in all challenging doses in case group was considerably higher than control groups (p < 0.05). On the other hand, the repair rate in those volunteers, who received less than 10.2 mSv/y was significantly more than the control groups. In the contrary, individuals who live in homes with more than 10.2 mSv/y had incomplete repair. Additionally the plasma and urinary levels of vitamin C were measured spectrophotometrically. Although the concentration of vitamin C of plasma was equal in case and control 1 groups, the urinary level of vitamin C was found to be lower in the case group.

  20. Origin and significance of the Permian high-K calc-alkaline magmatism in the central-eastern Southern Alps, Italy

    Science.gov (United States)

    Rottura, A.; Bargossi, G. M.; Caggianelli, A.; Del Moro, A.; Visonà, D.; Tranne, C. A.

    1998-12-01

    The Atesina Volcanic District, the Monte Luco volcanics, and the Cima d'Asta, Bressanone-Chiusa, Ivigna, Monte Croce and Monte Sabion intrusions, in the central-eastern Southern Alps, form a wide calc-alkaline association of Permian age (ca. 280-260 Ma). The magmatism originated during a period of post-orogenic extensional/transtensional faulting which controlled the magma ascent and emplacement. The magmatic products are represented by a continuum spectrum of rock types ranging from basaltic andesites to rhyolites, and from gabbros to monzogranites, with preponderance of the acidic terms. They constitute a metaluminous to weakly peraluminous series showing mineralogical, petrographic and chemical characteristics distinctive of the high-K calc-alkaline suites. In the MORB-normalized trace element diagrams, the most primitive volcanic and plutonic rocks (basaltic andesites and gabbros with Mg No.=66 to 70; Ni=25 to 83 ppm; Cr=248 to 679 ppm) show LILE and LREE enriched patterns with troughs at Nb-Ta and Ti, a distinctive feature of subduction-related magmas. Field, petrographic, geochemical and isotopic evidence (initial 87Sr/ 86Sr ratios from 0.7057 to 0.7114; ɛNd values from -2.7 to -7.4; ∂ 18O values between 7.6 and 9.5‰) support a hybrid nature for both volcanic and plutonic rocks, originating through complex interactions between mantle-derived magmas and crustal materials. Only the scanty andalusite-cordierite and orthopyroxene-cordierite bearing peraluminous granites in the Cima d'Asta and Bressanone-Chiusa intrusive complexes can be interpreted as purely crustal melts (initial 87Sr/ 86Sr=0.7143-0.7167; initial ɛNd values between -7.9 and -9.6, close to average composition of the granulitic metasedimentary crust from the Ivrea Zone in the western Southern Alps). Although the Permian magmatism shows geochemical characteristics similar to those of arc-related suites, palaeogeographic restorations, and geological and tectonic evidence, seem not to support

  1. Enhanced reductive dechlorination of tetrachloroethene by nano-sized mackinawite with cyanocobalamin in a highly alkaline condition.

    Science.gov (United States)

    Kim, Sangwoo; Park, Taehyung; Lee, Woojin

    2015-03-15

    In this study, we characterize the reductive dechlorination of tetrachloroethene (PCE) by nano-sized mackinawite (nFeS) with cobalamin (Cbl(III)) at a high pH and investigate the effects of environmental factors, including the concentrations of the target contaminant, reductant, and catalyst and suspension ions on the dechlorination kinetics of PCE. Ninety five percent of the PCE was degraded by nFeS with Cbl(III) in 15 h. Cyclic voltammetry conducted with regard to the reductive dechlorination showed a higher redox potential of mackinawite under a high-pH condition (-1.01 V), suggesting that the oxidation state of the central cobalt ion in the cobalamin could be reduced to Cbl(I). The change of cobalamin species on the nFeS surface was verified under different pH conditions by UV-vis spectroscopy. The rate constant of PCE dechlorination increased from 0.1582 to 0.4284 h(-1) due to the increase in the nFeS content (2.085-20.85 g/L). As the concentration of Cbl(III) increased from 0 to 0.5 mM, the dechlorination kinetics of PCE was accelerated (0-1.4091 h(-1)) but reached a state of equilibrium from 0.5 to 1 mM. The increase in the initial PCE concentration (0.035-1.0 mM) slowed down the dechlorination kinetics (0.2036-0.0962 h(-1)). The dechlorination kinetics was enhanced by 1.5-11 times when 10 mM of ions (Na(+), K(+), Mg(2+), Ca(2+), CO3(2-), SO4(2-), and NO3(-)) were added, while an addition of HCO3 decelerated it by 10 times. This study can provide background knowledge pertaining to the PCE dechlorination by a natural reductant under a high-pH condition and the effect of environmental factors on the dechlorination kinetics for the development of novel remediation technologies. Copyright © 2015 Elsevier Ltd. All rights reserved.

  2. High level extracellular production of a recombinant alkaline catalase in E. coli BL21 under ethanol stress and its application in hydrogen peroxide removal after cotton fabrics bleaching.

    Science.gov (United States)

    Yu, Zhenxiao; Zheng, Hongchen; Zhao, Xingya; Li, Shufang; Xu, Jianyong; Song, Hui

    2016-08-01

    The effects of induction parameters, osmolytes and ethanol stress on the productivity of the recombinant alkaline catalase (KatA) in Escherichia coli BL21 (pET26b-KatA) were investigated. The yield of soluble KatA was significantly enhanced by 2% ethanol stress. And a certain amount of Triton X-100 supplementation could markedly improved extracellular ratio of KatA. A total soluble catalase activity of 78,762U/mL with the extracellular ratio of 92.5% was achieved by fed-batch fermentation in a 10L fermentor, which was the highest yield so far. The purified KatA showed high stability at 50°C and pH 6-10. Application of KatA for elimination of H2O2 after cotton fabrics bleaching led to less consumption of water, steam and electric power by 25%, 12% and 16.7% respectively without productivity and quality losing of cotton fabrics. Thus, the recombinant KatA is a promising candidate for industrial production and applications.

  3. Fever of unknown origin (FUO) due to large B-cell lymphoma: the diagnostic significance of highly elevated alkaline phosphatase and serum ferritin levels.

    Science.gov (United States)

    Cunha, Burke A; Petelin, Andrew

    2013-01-01

    Determining the cause of fever of unknown origin (FUO) is often a vexing and difficult diagnostic process. In most cases, the signs and symptoms in adult FUOs suggest a malignant, infectious, or rheumatic/inflammatory etiology. The diagnosis of FUO may be narrowed if specific findings are present (eg, hepatosplenomegaly) that limit the diagnostic possibilities. Infectious causes of FUO with hepatosplenomegaly include miliary tuberculosis, typhoid fever, and visceral leishmanosis (kala-azar). However, FUOs with hepatosplenomegaly are most often attributable to malignant neoplasms, ie, Hodgkin lymphoma, non-Hodgkin lymphoma, hepatoma, hypernephroma (renal-cell carcinoma), or preleukemia. We present a middle-aged woman with FUO and hepatosplenomegaly. Inpatient nonspecific laboratory findings included a highly elevated erythrocyte sedimentation rate, and elevated levels of vitamin B12, lactate dehydrogenase, angiotensin-converting enzyme, ferritin, and alkaline phosphatase. These individual findings are nonspecific, but together point to a lymphoma. An important test in differentiating malignant from infectious FUOs is the Naprosyn test, and her Naprosyn test was positive, indicating malignancy. A gallium scan suggested a uterine lymphoma. A computed tomography scan revealed hepatosplenomegaly, but the gallium uptake was not increased in her liver and spleen. Uterine and bone marrow biopsies were negative for lymphoma. We present a case of FUO with hepatosplenomegaly attributable to large B-cell lymphoma as diagnosed via liver biopsy. Copyright © 2013 Elsevier Inc. All rights reserved.

  4. [High current microsecond pulsed hollow cathode lamp excited ionic fluorescence spectrometry of alkaline earth elements in inductively coupled plasma with a Fassel-torch].

    Science.gov (United States)

    Zhang, Shao-Yu; Gong, Zhen-Bin; Huang, Ben-Li

    2006-02-01

    High current microsecond pulsed hollow cathode lamp (HCMP-HCL) excited ionic fluorescence spectrometry (IFS) of alkaline earth elements in inductively coupled plasma (ICP) with a Fassel-torch has been investigated. In wide condition ranges only IFS was observed, whilst atomic fluorescence spectrometry (AFS) was not detectable. More intense ionic fluorescence signal was observed at lower observation heights and at lower incident RF powers. Without introduction of any reduction organic gases into the ICP, the limit of detection (LOD, 3sigma) of Ba was improved by 50-fold over that of a conventional pulsed (CP) HCL with the Baird sleeve-extended torch. For Ca and Sr, the LODs by HCMP-HCL-ICP-IFS and CP-HCL-ICP-AFS show no significant difference. Relative standard deviations were 0.6%-1.4% (0.1-0.2 microg x mL(-1), n = 10) for 5 ionic fluorescence lines. Preliminary studies showed that the intensity of ionic fluorescence could be depressed in the presence of K, Al and P.

  5. The family of Deg/HtrA proteases in plants

    Directory of Open Access Journals (Sweden)

    Schuhmann Holger

    2012-04-01

    Full Text Available Abstract Background The Deg/HtrA family of ATP-independent serine endopeptidases is present in nearly all organisms from bacteria to human and vascular plants. In recent years, multiple deg/htrA protease genes were identified in various plant genomes. During genome annotations most proteases were named according to the order of discovery, hence the same names were sometimes given to different types of Deg/HtrA enzymes in different plant species. This can easily lead to false inference of individual protease functions based solely on a shared name. Therefore, the existing names and classification of these proteolytic enzymes does not meet our current needs and a phylogeny-based standardized nomenclature is required. Results Using phylogenetic and domain arrangement analysis, we improved the nomenclature of the Deg/HtrA protease family, standardized protease names based on their well-established nomenclature in Arabidopsis thaliana, and clarified the evolutionary relationship between orthologous enzymes from various photosynthetic organisms across several divergent systematic groups, including dicots, a monocot, a moss and a green alga. Furthermore, we identified a “core set” of eight proteases shared by all organisms examined here that might provide all the proteolytic potential of Deg/HtrA proteases necessary for a hypothetical plant cell. Conclusions In our proposed nomenclature, the evolutionarily closest orthologs have the same protease name, simplifying scientific communication when comparing different plant species and allowing for more reliable inference of protease functions. Further, we proposed that the high number of Deg/HtrA proteases in plants is mainly due to gene duplications unique to the respective organism.

  6. Cross genome comparisons of serine proteases in Arabidopsis and rice

    Directory of Open Access Journals (Sweden)

    Sowdhamini R

    2006-08-01

    Full Text Available Abstract Background Serine proteases are one of the largest groups of proteolytic enzymes found across all kingdoms of life and are associated with several essential physiological pathways. The availability of Arabidopsis thaliana and rice (Oryza sativa genome sequences has permitted the identification and comparison of the repertoire of serine protease-like proteins in the two plant species. Results Despite the differences in genome sizes between Arabidopsis and rice, we identified a very similar number of serine protease-like proteins in the two plant species (206 and 222, respectively. Nearly 40% of the above sequences were identified as potential orthologues. Atypical members could be identified in the plant genomes for Deg, Clp, Lon, rhomboid proteases and species-specific members were observed for the highly populated subtilisin and serine carboxypeptidase families suggesting multiple lateral gene transfers. DegP proteases, prolyl oligopeptidases, Clp proteases and rhomboids share a significantly higher percentage orthology between the two genomes indicating substantial evolutionary divergence was set prior to speciation. Single domain architectures and paralogues for several putative subtilisins, serine carboxypeptidases and rhomboids suggest they may have been recruited for additional roles in secondary metabolism with spatial and temporal regulation. The analysis reveals some domain architectures unique to either or both of the plant species and some inactive proteases, like in rhomboids and Clp proteases, which could be involved in chaperone function. Conclusion The systematic analysis of the serine protease-like proteins in the two plant species has provided some insight into the possible functional associations of previously uncharacterised serine protease-like proteins. Further investigation of these aspects may prove beneficial in our understanding of similar processes in commercially significant crop plant species.

  7. Alkaline earth metal thioindates

    Energy Technology Data Exchange (ETDEWEB)

    Ivanov-Ehmin, B.N.; Ivlieva, V.I.; Filatenko, L.A.; Zajtsev, B.E.; Kaziev, G.Z.; Sarabiya, M.G.

    1984-08-01

    Alkaline earth metal thioindates of MIn/sub 2/S/sub 4/ composition were synthesized by interaction of alkaline earth metal oxoindates with hydrogen sulfide during heating. Investigation into the compounds by X-ray analysis showed that calcium compound crystallizes in cubic crystal system and strontium and barium compounds in rhombic crystal system. Lattice parameters and the number of formula units were determined. Thioindates of M/sub 3/In/sub 2/S/sub 6/ composition were synthesized, their individuality was shown.

  8. Specific and efficient cleavage of fusion proteins by recombinant plum pox virus NIa protease.

    Science.gov (United States)

    Zheng, Nuoyan; Pérez, José de Jesús; Zhang, Zhonghui; Domínguez, Elvira; Garcia, Juan Antonio; Xie, Qi

    2008-02-01

    Site-specific proteases are the most popular kind of enzymes for removing the fusion tags from fused target proteins. Nuclear inclusion protein a (NIa) proteases obtained from the family Potyviridae have become promising due to their high activities and stringencies of sequences recognition. NIa proteases from tobacco etch virus (TEV) and tomato vein mottling virus (TVMV) have been shown to process recombinant proteins successfully in vitro. In this report, recombinant PPV (plum pox virus) NIa protease was employed to process fusion proteins with artificial cleavage site in vitro. Characteristics such as catalytic ability and affecting factors (salt, temperature, protease inhibitors, detergents, and denaturing reagents) were investigated. Recombinant PPV NIa protease expressed and purified from Escherichia coli demonstrated efficient and specific processing of recombinant GFP and SARS-CoV nucleocapsid protein, with site F (N V V V H Q black triangle down A) for PPV NIa protease artificially inserted between the fusion tags and the target proteins. Its catalytic capability is similar to those of TVMV and TEV NIa protease. Recombinant PPV NIa protease reached its maximal proteolytic activity at approximately 30 degrees C. Salt concentration and only one of the tested protease inhibitors had minor influences on the proteolytic activity of PPV NIa protease. Recombinant PPV NIa protease was resistant to self-lysis for at least five days.

  9. Influence of Mn-dopant on the properties of {alpha}-FeOOH particles precipitated in highly alkaline media

    Energy Technology Data Exchange (ETDEWEB)

    Krehula, Stjepko [Division of Materials Chemistry, Ruder Boskovic Institute, P.O. Box 180, HR-10002 Zagreb (Croatia); Music, Svetozar [Division of Materials Chemistry, Ruder Boskovic Institute, P.O. Box 180, HR-10002 Zagreb (Croatia)]. E-mail: music@irb.hr

    2006-12-21

    The effects of Mn-dopant on the formation of solid solutions {alpha}-(Fe, Mn)OOH in dependence on the initial concentration ratio r = [Mn]/([Mn] + [Fe]), as well as on the size and morphology of the corresponding particles were investigated using Moessbauer and FT-IR spectroscopies, high-resolution scanning electron microscopy (FE SEM) and an energy dispersive X-ray analyser (EDS). The value of the hyperfine magnetic field of 34.9 T, as recorded for the reference {alpha}-FeOOH sample at RT, decreased linearly up to 21.4 T for sample with r = 0.1667. Only a paramagnetic doublet at RT was recorded for sample with r = 0.2308, a ferrite phase was additionally found for r = 0.3333. Fe-OH bending IR bands, {delta} {sub OH} and {gamma} {sub OH}, were influenced by the Mn-substitution as manifested through their gradual shifts. FE SEM micrographs showed a great elongation of the starting acicular particles along the c-axis with an increase in Mn-doping. For r = 0.1667 and 0.2308 star-shaped and dendritic twin {alpha}-(Fe, Mn)OOH particles were observed. The length of these {alpha}-(Fe, Mn)OOH particles decreased, whereas their width increased. The {alpha}-Fe{sub 2}O{sub 3} phase was not detected in any of the samples prepared.

  10. High frequencies of elevated alkaline phosphatase activity and rickets exist in extremely low birth weight infants despite current nutritional support

    Directory of Open Access Journals (Sweden)

    Parker Bruce R

    2009-07-01

    Full Text Available Abstract Background Osteopenia and rickets are common among extremely low birth weight infants (ELBW, Methods We evaluated all ELBW infants admitted to Texas Children's Hospital NICU in 2006 and 2007. Of 211 admissions, we excluded 98 patients who were admitted at >30 days of age or did not survive/stay for >6 weeks. Bone radiographs obtained in 32 infants were reviewed by a radiologist masked to laboratory values. Results In this cohort of 113 infants, P-APA was found to have a significant inverse relationship with BW, gestational age and serum phosphorus. In paired comparisons, P-APA of infants Conclusion Elevation of P-APA >600 IU/L was very common in ELBW infants. BW was significantly inversely related to both P-APA and radiologic rickets. No single value of P-APA was related to radiological findings of rickets. Given the very high risk of osteopenia and rickets among ELBW infants, we recommend consideration of early screening and early mineral supplementation, especially among infants

  11. Characterization and expression levels of protease enzymes at different developmental stages of Siamese fighting fish (Betta splendens Regan, 1910)

    OpenAIRE

    Thongprajukaew, Karun; Kovitvadhi, Uthaiwan; Engkagul, Arunee; Torrissen, Krisna Rungruangsak

    2010-01-01

    Characterization of total proteases, trypsin and chymotrypsin were performed at pH 2 12 and temperatures 20-80°C in three life stages of Siamese fighting fish (Betta splendens Regan, 1910). At least fourteen protease activities were detected, including seven acidic (pH levels 2, 4 and 5-6), one neutral (pH 7), and six alkaline (pH levels 8-12) activities in the optimal temperature range of 30-60°C. The neutral and alkaline pH profiles were different between sexes during the on-gro...

  12. Exploring a new serine protease from Cucumis sativus L.

    Science.gov (United States)

    Nafeesa, Zohara; Shivalingu, B R; Vivek, H K; Priya, B S; Swamy, S Nanjunda

    2015-03-01

    Coagulation is an important physiological process in hemostasis which is activated by sequential action of proteases. This study aims to understand the involvement of aqueous fruit extract of Cucumis sativus L. (AqFEC) European burp less variety in blood coagulation cascade. AqFEC hydrolyzed casein in a dose-dependent manner. The presence of protease activity was further confirmed by casein zymography which revealed the possible presence of two high molecular weight protease(s). The proteolytic activity was inhibited only by phenyl methyl sulphonyl fluoride suggesting the presence of serine protease(s). In a dose-dependent manner, AqFEC also hydrolysed Aα and Bβ subunits of fibrinogen, whereas it failed to degrade the γ subunit of fibrinogen even at a concentration as high as 100 μg and incubation time up to 4 h. AqFEC reduced the clotting time of citrated plasma by 87.65%. The protease and fibrinogenolytic activity of AqFEC suggests its possible role in stopping the bleeding and ensuing wound healing process.

  13. Cold-adapted proteases as an emerging class of therapeutics.

    Science.gov (United States)

    Fornbacke, Marcus; Clarsund, Mats

    2013-06-01

    Proteases have been used in medicine for several decades and are an established and well tolerated class of therapeutic agent. These proteases were sourced from mammals or bacteria that exist or have adapted to moderate temperatures (mesophilic organisms); however, proteases derived from organisms from cold environments-cold-adapted or psychrophilic proteases-generally have high specific activity, low substrate affinity, and high catalytic rates at low and moderate temperatures. Made possible by greater flexibility, psychrophilic enzymes interact with and transform the substrate at lower energy costs. Cold-adapted proteases have been used in a wide range of applications, including industrial functions, textiles, cleaning/hygiene products, molecular biology, environmental bioremediations, consumer food products, cosmetics, and pharmaceutical production. In addition to these applications, they have also shown promise as therapeutic modalities for cosmeceutical applications (by reducing glabellar [frown] lines) and a number of disease conditions, including bacterial infections (by disrupting biofilms to prevent bacterial infection), topical wound management (when used as a debridement agent to remove necrotic tissue and fibrin clots), oral/dental health management (by removing plaque and preventing periodontal disease), and in viral infections (by reducing the infectivity of viruses, such as human rhinovirus 16 and herpes simplex virus). Psychrophilic proteases with greater activity and stability (than the original organism-derived variant) have been developed; this coupled with available manufacturing recombinant production techniques suggests that cold-adapted proteases have a promising future as a distinct therapeutic class with diverse clinical applications.

  14. [Advances of alkaline amylase production and applications].

    Science.gov (United States)

    Yang, Haiquan; Liu, Long; Li, Jianghua; Du, Guocheng; Chen, Jian

    2012-04-01

    Alkaline amylase is one of alkaline enzymes with optimum pH in the alkaline range, and it could keep stability and efficiently hydrolyze starch under alkaline conditions. Alkaline amylase finds wide applications in textile, detergent, pharmaceutical, food and other fields. Alkaline amylases could be produced by alkaliphilic microorganisms. In this work, the advances of alkaline amylase production and applications were reviewed.

  15. PROTEASES AND PROTEASE INHIBITORS INTERACTION: DEFENCE STRATEGY AGAINST

    Directory of Open Access Journals (Sweden)

    R.S.DHANDE 1 N.J.CHIKHALE 2

    2014-12-01

    Full Text Available ABSTRACT: An increase in crop yield, its management and preservation are among the main challenges standing before the human population that exceed 10 billion by the mid of 21 st  century.  Every year, considerable agricultural losses occur due to repeated practices of cultivation of large genetically similar populations.  Such cultivation practices favors incidence of more insect pests (Hilder and Boulter, 1999;  Oerke  et  al.,  1994;  Smith,  1999.  To  solve  these  problems,  current approaches  rely  on  use  of  synthetic  chemicals  like  fertilizers,  insecticides, herbicides,  fungicides  etc.  But  this  exerts  excessively  high  pressure  on environment  and  destabilizes  the  ecological  balance.  The  traditional  pest control method involves the use of conventional pesticides, most of which are non-specific and wipe out the entire community, pollutes the agro-ecosystem, and  increases  the  cost  of  production.  The  emergence  of  gene  transfer technology  has  solved  some  problems  regarding  overuse  of  chemical pesticides.  The  delta  endotoxin  encoding  gene  from  Bacillus  thuringiensis,  a gram positive soil borne bacteria transferred in crops has given little relief from coleopterans and lepidopterans attack.  Whereas, the insects belonging to these orders like Helicoverpa Sps. have developed resistance against Bt toxins. The other approach takes advantage of use of plant genes encoding defense proteins like protease inhibitors which is more appealing, simpler and safer (Dunaevsky et.  al.,  2005.  Proteinase  inhibitors  (PIs  are  naturally  occurring  proteins  in living organisms and are able to inhibit & control the activity of proteases. PIs act  on  an  active  site  of  digestive  proteolytic  enzymes  and  form  a  stable complex  unlike  enzyme-substrate  or  enzyme-product  weak  complexes  which

  16. Alkaline Capacitors Based on Nitride Nanoparticles

    Science.gov (United States)

    Aldissi, Matt

    2003-01-01

    High-energy-density alkaline electrochemical capacitors based on electrodes made of transition-metal nitride nanoparticles are undergoing development. Transition- metal nitrides (in particular, Fe3N and TiN) offer a desirable combination of high electrical conductivity and electrochemical stability in aqueous alkaline electrolytes like KOH. The high energy densities of these capacitors are attributable mainly to their high capacitance densities, which, in turn, are attributable mainly to the large specific surface areas of the electrode nanoparticles. Capacitors of this type could be useful as energy-storage components in such diverse equipment as digital communication systems, implanted medical devices, computers, portable consumer electronic devices, and electric vehicles.

  17. Photo-Fenton oxidation of phenol and organochlorides (2,4-DCP and 2,4-D) in aqueous alkaline medium with high chloride concentration.

    Science.gov (United States)

    Luna, Airton J; Chiavone-Filho, Osvaldo; Machulek, Amilcar; de Moraes, José Ermírio F; Nascimento, Cláudio A O

    2012-11-30

    A highly concentrated aqueous saline-containing solution of phenol, 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4-dichlorophenol (2,4-DCP) was treated by the photo-Fenton process in a system composed of an annular reactor with a quartz immersion well and a medium-pressure mercury lamp (450 W). The study was conducted under special conditions to minimize the costs of acidification and neutralization, which are usual steps in this type of process. Photochemical reactions were carried out to investigate the influence of some process variables such as the initial concentration of Fe(2+) ([Fe(2+)](0)) from 1.0 up to 2.5 mM, the rate in mmol of H(2)O(2) fed into the system (FH(2)O(2);in) from 3.67 up to 7.33 mmol of H(2)O(2)/min during 120 min of reaction time, and the initial pH (pH(0)) from 3.0 up to 9.0 in the presence and absence of NaCl (60.0 g/L). Although the optimum pH for the photo-Fenton process is about 3.0, this particular system performed well in experimental conditions starting at alkaline and neutral pH. The results obtained here are promising for industrial applications, particularly in view of the high concentration of chloride, a known hydroxyl radical scavenger and the main oxidant present in photo-Fenton processes.

  18. Allergens with Protease Activity from House Dust Mites

    Directory of Open Access Journals (Sweden)

    Manuel Reithofer

    2017-06-01

    Full Text Available Globally, house dust mites (HDM are one of the main sources of allergens causing Type I allergy, which has a high risk of progressing into a severe disabling disease manifestation such as allergic asthma. The strong protease activities of a number of these allergens are thought to be involved in several steps of the pathophysiology of this allergic disease. It has been a common notion that protease activity may be one of the properties that confers allergenicity to proteins. In this review we summarize and discuss the roles of the different HDM proteases in the development of Type I allergy.

  19. The S8 serine, C1A cysteine and A1 aspartic protease families in Arabidopsis.

    Science.gov (United States)

    Beers, Eric P; Jones, Alan M; Dickerman, Allan W

    2004-01-01

    The Arabidopsis thaliana genome has over 550 protease sequences representing all five catalytic types: serine, cysteine, aspartic acid, metallo and threonine (MEROPS peptidase database, http://merops.sanger.ac.uk/), which probably reflect a wide variety of as yet unidentified functions performed by plant proteases. Recent indications that the 26S proteasome, a T1 family-threonine protease, is a regulator of light and hormone responsive signal transduction highlight the potential of proteases to participate in many aspects of plant growth and development. Recent discoveries that proteases are required for stomatal distribution, embryo development and disease resistance point to wider roles for four additional multigene families that include some of the most frequently studied (yet poorly understood) plant proteases: the subtilisin-like, serine proteases (family S8), the papain-like, cysteine proteases (family C1A), the pepsin-like, aspartic proteases (family A1) and the plant matrixin, metalloproteases (family M10A). In this report, 54 subtilisin-like, 30 papain-like and 59 pepsin-like proteases from Arabidopsis, are compared with S8, C1A and A1 proteases known from other plant species at the functional, phylogenetic and gene structure levels. Examples of structural conservation between S8, C1A and A1 genes from rice, barley, tomato and soybean and those from Arabidopsis are noted, indicating that some common, essential plant protease roles were established before the divergence of monocots and eudicots. Numerous examples of tandem duplications of protease genes and evidence for a variety of restricted expression patterns suggest that a high degree of specialization exists among proteases within each family. We propose that comprehensive analysis of the functions of these genes in Arabidopsis will firmly establish serine, cysteine and aspartic proteases as regulators and effectors of a wide range of plant processes.

  20. Purification and characterization of a unique alkaline elastase from Micrococcus luteus.

    Science.gov (United States)

    Clark, D J; Hawrylik, S J; Kavanagh, E; Opheim, D J

    2000-02-01

    Micrococcus luteus isolated from human skin secretes an alkaline protease which degrades elastin. M. luteus protease (MLP) was produced in the late logarithmic and stationary phases of growth. MLP, purified to homogeneity by a three-step process, had a molecular mass of 32,812 Da and an isoelectric point of 9.3. MLP was active and highly stable in solution for 24 h from pH 6.0 to 10.5; it had maximal activity at temperatures between 57 and 59 degrees C. The presence of calcium in the solution was essential for enzyme activity and to prevent autolysis. Optimal activity occurred between pH 9.0 and 9.5, with 60% maximal activity from pH 6.5 to 11.0. The enzyme was inhibited by the serine enzyme inhibitors phenylmethylsulfonyl fluoride and chymostatin but not by the metalloenzyme inhibitor 1,10-phenanthroline or sulfhydryl enzyme inhibitors. Casein, bovine serum albumin, ovalbumin, beta-lactoglobulin, and elastin were digested by the protease while collagen and keratin were resistant to digestion. MLP demonstrated both esterase and amidase activity on synthetic peptide substrates. MLP preferentially cleaved the Leu(15)-Tyr(16) and Phe(24)-Phe(25) bonds of the oxidized beta-chain of insulin. Longer digests of insulin and the pattern of activity against synthetic substrates suggest that MLP has a cleavage specificity for bulky, hydrophobic, or aromatic amino acids in the P(1) or P(1)' positions. Amino acid sequences from the N-terminus and internal peptides of MLP were unique.

  1. In-cell protease assay systems based on trans-localizing molecular beacon proteins using HCV protease as a model system.

    Directory of Open Access Journals (Sweden)

    Jeong Hee Kim

    Full Text Available This study describes a sensitive in-cell protease detection system that enables direct fluorescence detection of a target protease and its inhibition inside living cells. This live-cell imaging system provides a fluorescent molecular beacon protein comprised of an intracellular translocation signal sequence, a protease-specific cleavage sequence, and a fluorescent tag sequence(s. The molecular beacon protein is designed to change its intracellular localization upon cleavage by a target protease, i.e., from the cytosol to a subcellular organelle or from a subcellular organelle to the cytosol. Protease activity can be monitored at the single cell level, and accordingly the entire cell population expressing the protease can be accurately enumerated. The clear cellular change in fluorescence pattern makes this system an ideal tool for various life science and drug discovery research, including high throughput and high content screening applications.

  2. Fullerene-C60 and crown ether doped on C60 sensors for high sensitive detection of alkali and alkaline earth cations

    Science.gov (United States)

    Zaghmarzi, Fatemeh Alipour; Zahedi, Mansour; Mola, Adeleh; Abedini, Saboora; Arshadi, Sattar; Ahmadzadeh, Saeed; Etminan, Nazanin; Younesi, Omran; Rahmanifar, Elham; Yoosefian, Mehdi

    2017-03-01

    Fullerenes are effective acceptor components with high electron affinity for charge transfer. The significant influences of chemical adsorption of the cations on the electrical sensitivity of pristine C60 and 15-(C2H4O)5/C60 nanocages could be the basis of new generation of electronic sensor design. The density functional theory calculation for alkali and alkaline earth cations detection by pristine C60 and 15-(C2H4O)5/C60 nanocages are considered at B3LYP level of theory with 6-31 G(d) basis set. The quantum theory of atoms in molecules analysis have been performed to understand the nature of intermolecular interactions between the cations and nanocages. Also, the natural bond orbital analysis have been performed to assess the intermolecular interactions in detail. Furthermore, the frontier molecular orbital, energy gap, work function, electronegativity, number of transferred electron (∆N), dipole moment as well as the related chemical hardness and softness are investigated and calculated in this study. The results show that the adsorption of cations (M=Na+, K+, Mg2+ and Ca2+) are exothermic and the binding energy in pristine C60 nanocage and 15-(C2H4O)5/C60 increases with respect to the cations charge. The results also denote a decrease in the energy gap and an increase in the electrical conductivity upon the adsorption process. In order to validate the obtained results, the density of state calculations are employed and presented in the end as well.

  3. Two 3D structured Co-Ni bimetallic oxides as cathode catalysts for high-performance alkaline direct methanol fuel cells

    Science.gov (United States)

    Liu, Yan; Shu, Chengyong; Fang, Yuan; Chen, Yuanzhen; Liu, Yongning

    2017-09-01

    Two NiCo2O4 bimetallic oxides were synthesized via a facile hydrothermal method. SEM and TEM observations show that these materials have three-dimensional (3D) dandelion-like (DL) and flower-like (FL) morphologies. Their large specific surface areas (90.68 and 19.8 m2·g-1) and porous structures provide many active sites and effective transport pathways for the oxygen reduction reaction (ORR). Electrochemical measurements with a rotating ring-disc electrode (RRDE) indicate that the electron transfer numbers of the NiCo2O4-DL and NiCo2O4-FL catalysts for ORR in an alkaline solution are 3.97 and 3.91, respectively. Fuel cells were assembled with the bimetallic oxides, PtRu/C and a polymer fiber membrane (PFM) as cathode catalysts, anode catalyst and electrolyte film, respectively. For NiCo2O4-DL, the peak power density reaches up to 73.5 mW·cm-2 at 26 °C, which is the highest room-temperature value reported to date. The high catalytic activity of NiCo2O4 is mainly attributed to the presence of many Co3+ cations that directly donate electrons to O2 to reduce it via a more efficient and effective route. Furthermore, the catalytic performance of NiCo2O4-DL is superior to that of NiCo2O4-FL because it has a higher specific surface area and is less crystalline.

  4. The Anfeg post-collisional Pan-African high-K calc-alkaline batholith (Central Hoggar, Algeria), result of the LATEA microcontinent metacratonization

    Science.gov (United States)

    Acef, Kaissa; Liégeois, Jean Paul; Ouabadi, Aziouz; Latouche, Louis

    2003-10-01

    The Anfeg batholith (or composite laccolith) occupies a large surface (2000 km 2) at the northern tip of the Laouni terrane, just south of Tamanrasset in Hoggar. It is granodioritic to granitic in composition and comprises abundant enclaves that are either mafic microgranular enclaves (MME) or gneissic xenoliths. It intruded an Eburnian (≈2 Ga) high-grade basement belonging to the LATEA metacraton at approximately 608 Ma (recalculated from the U-Pb dating of [Tectonics 5 (1986) 955]) and cooled at approximately 4 kbar, with a temperature of about 750 °C. This emplacement occurred mainly along subhorizontal thrust planes related to Pan-African subvertical mega-shear zones close to the attachment zone of a strike-slip partitioned transpression system. Although affected by some LILE mobility, the Anfeg batholith can be ascribed to a high-K calc-alkaline suite but characterized by low heavy REE contents and high LREE/HREE ratios. The MME belong to the Anfeg magmatic trend while some xenoliths belong to Neoproterozoic island arc rocks. The Anfeg batholith defines a Nd-Sr isotopic initial ratios trend ( ɛNd/( 87Sr/ 86Sr) i from -2.8/0.7068 to -11.8/0.7111) pointing to a mixing between a depleted mantle and an old Rb-depleted granulitic lower crust. Both sources have been identified within LATEA and elsewhere in the Tuareg shield ( ɛNd/ 87Sr/ 86Sr) i of +6.2/0.7028 for the depleted mantle, -22/0.708 for the old lower crust. The model proposed relates the above geochemical features to a lithospheric delamination along the subvertical mega-shear zones that dissected the rigid LATEA former passive margin without major crustal thickening (metacratonization) during the general northward tectonic escape of the Tuareg terranes, a consequence of the collision with the West African craton. This delamination allowed the uprise of the asthenosphere. In turn, this induced the melting of the asthenosphere by adiabatic pressure release and of the old felsic and mafic lower crust

  5. Cloning, Expression and Activity Analysis of a Novel Fibrinolytic Serine Protease fromArenicola cristata

    Institute of Scientific and Technical Information of China (English)

    ZHAO Chunling; JU Jiyu

    2015-01-01

    The full-length cDNA of a protease gene from a marine annelid Arenicola cristata was amplified through rapid amplifi-cation of cDNA ends technique and sequenced. The size of the cDNA was 936 bp in length, including an open reading frame encod-ing a polypeptide of 270 amino acid residues. The deduced amino acid sequnce consisted of pro- and mature sequences. The protease belonged to the serine protease family because it contained the highly conserved sequence GDSGGP. This protease was novel as it showed a low amino acid sequence similarity (<40%) to other serine proteases. The gene encoding the active form ofA. cristata serine protease was cloned and expressed inE. coli. Purified recombinant protease in a supernatant could dissolve an artificial fibrin plate with plasminogen-rich fibrin, whereas the plasminogen-free fibrin showed no clear zone caused by hydrolysis. This result sug-gested that the recombinant protease showed an indirect fibrinolytic activity of dissolving fibrin, and was probably a plasminogen activator. A rat model with venous thrombosis was established to demonstrate that the recombinant protease could also hydrolyze blood clotin vivo. Therefore, this recombinant protease may be used as a thrombolytic agent for thrombosis treatment. To our knowledge, this study is the first of reporting the fibrinolytic serine protease gene inA. cristata.

  6. Cloning, expression and activity analysis of a novel fibrinolytic serine protease from Arenicola cristata

    Science.gov (United States)

    Zhao, Chunling; Ju, Jiyu

    2015-06-01

    The full-length cDNA of a protease gene from a marine annelid Arenicola cristata was amplified through rapid amplification of cDNA ends technique and sequenced. The size of the cDNA was 936 bp in length, including an open reading frame encoding a polypeptide of 270 amino acid residues. The deduced amino acid sequnce consisted of pro- and mature sequences. The protease belonged to the serine protease family because it contained the highly conserved sequence GDSGGP. This protease was novel as it showed a low amino acid sequence similarity (< 40%) to other serine proteases. The gene encoding the active form of A. cristata serine protease was cloned and expressed in E. coli. Purified recombinant protease in a supernatant could dissolve an artificial fibrin plate with plasminogen-rich fibrin, whereas the plasminogen-free fibrin showed no clear zone caused by hydrolysis. This result suggested that the recombinant protease showed an indirect fibrinolytic activity of dissolving fibrin, and was probably a plasminogen activator. A rat model with venous thrombosis was established to demonstrate that the recombinant protease could also hydrolyze blood clot in vivo. Therefore, this recombinant protease may be used as a thrombolytic agent for thrombosis treatment. To our knowledge, this study is the first of reporting the fibrinolytic serine protease gene in A. cristata.

  7. Proteases of Wood Rot Fungi with Emphasis on the Genus Pleurotus

    Directory of Open Access Journals (Sweden)

    Fabíola Dorneles Inácio

    2015-01-01

    Full Text Available Proteases are present in all living organisms and they play an important role in physiological conditions. Cell growth and death, blood clotting, and immune defense are all examples of the importance of proteases in maintaining homeostasis. There is growing interest in proteases due to their use for industrial purposes. The search for proteases with specific characteristics is designed to reduce production costs and to find suitable properties for certain industrial sectors, as well as good producing organisms. Ninety percent of commercialized proteases are obtained from microbial sources and proteases from macromycetes have recently gained prominence in the search for new enzymes with specific characteristics. The production of proteases from saprophytic basidiomycetes has led to the identification of various classes of proteases. The genus Pleurotus has been extensively studied because of its ligninolytic enzymes. The characteristics of this genus are easy cultivation techniques, high yield, low nutrient requirements, and excellent adaptation. There are few studies in the literature about proteases of Pleurotus spp. This review gathers together information about proteases, especially those derived from basidiomycetes, and aims at stimulating further research about fungal proteases because of their physiological importance and their application in various industries such as biotechnology and medicine.

  8. Cathepsin proteases in Toxoplasma gondii

    Science.gov (United States)

    Dou, Zhicheng; Carruthers, Vern B.

    2014-01-01

    Cysteine proteases are important for the growth and survival of apicomplexan parasites that infect humans. The apicomplexan Toxoplasma gondii expresses five members of the C1 family of cysteine proteases, including one cathepsin L-like (TgCPL), one cathepsin B-like (TgCPB), and three cathepsin C-like (TgCPC1, 2 and 3) proteases. Recent genetic, biochemical and structural studies reveal that cathepsins function in microneme and rhoptry protein maturation, host cell invasion, replication, and nutrient acquisition.. Here, we review the key features and roles of T. gondii cathepsins and discuss the therapeutic potential for specific inhibitor development. PMID:21660658

  9. Alkaline broadening in Stars

    CERN Document Server

    De Kertanguy, A

    2015-01-01

    Giving new insight for line broadening theory for atoms with more structure than hydrogen in most stars. Using symbolic software to build precise wave functions corrected for ds;dp quantum defects. The profiles obtained with that approach, have peculiar trends, narrower than hydrogen, all quantum defects used are taken from atomic database topbase. Illustration of stronger effects of ions and electrons on the alkaline profiles, than neutral-neutral collision mechanism. Keywords : Stars: fundamental parameters - Atomic processes - Line: profiles.

  10. A novel dengue virus inhibitor, BP13944, discovered by high-throughput screening with dengue virus replicon cells selects for resistance in the viral NS2B/NS3 protease.

    Science.gov (United States)

    Yang, Chi-Chen; Hu, Han-Shu; Wu, Ren-Huang; Wu, Szu-Huei; Lee, Shiow-Ju; Jiaang, Weir-Torn; Chern, Jyh-Haur; Huang, Zhi-Shun; Wu, Huey-Nan; Chang, Chung-Ming; Yueh, Andrew

    2014-01-01

    Dengue virus (DENV) causes disease globally, resulting in an estimated 25 to 100 million new infections per year. No effective DENV vaccine is available, and the current treatment is only supportive. Thus, there is an urgent need to develop therapeutic agents to cure this epidemic disease. In the present study, we identified a potential small-molecule inhibitor, BP13944, via high-throughput screening (HTS) of 60,000 compounds using a stable cell line harboring an efficient luciferase replicon of DENV serotype 2 (DENV-2). BP13944 reduced the expression of the DENV replicon reporter in cells, showing a 50% effective concentration (EC50) of 1.03 ± 0.09 μM. Without detectable cytotoxicity, the compound inhibited replication or viral RNA synthesis in all four serotypes of DENV but not in Japanese encephalitis virus (JEV). Sequencing analyses of several individual clones derived from BP13944-resistant RNAs purified from cells harboring the DENV-2 replicon revealed a consensus amino acid substitution (E66G) in the region of the NS3 protease domain. Introduction of E66G into the DENV replicon, an infectious DENV cDNA clone, and recombinant NS2B/NS3 protease constructs conferred 15.2-, 17.2-, and 3.1-fold resistance to BP13944, respectively. Our results identify an effective small-molecule inhibitor, BP13944, which likely targets the DENV NS3 protease. BP13944 could be considered part of a more effective treatment regime for inhibiting DENV in the future.

  11. Mitochondrial cereblon functions as a Lon-type protease

    OpenAIRE

    Kosuke Kataoka; China Nakamura; Toru Asahi; Naoya Sawamura

    2016-01-01

    Lon protease plays a major role in the protein quality control system in mammalian cell mitochondria. It is present in the mitochondrial matrix, and degrades oxidized and misfolded proteins, thereby protecting the cell from various extracellular stresses, including oxidative stress. The intellectual disability-associated and thalidomide-binding protein cereblon (CRBN) contains a large, highly conserved Lon domain. However, whether CRBN has Lon protease-like function remains unknown. Here, we ...

  12. Optimization Conditions of Extracellular Proteases Production from a Newly Isolated Streptomyces Pseudogrisiolus NRC-15

    Directory of Open Access Journals (Sweden)

    El-Sayed E. Mostafa

    2012-01-01

    Full Text Available Microbial protease represents the most important industrial enzymes, which have an active role in biotechnological processes. The objective of this study was to isolate new strain of Streptomyces that produce proteolytic enzymes with novel properties and the development of the low-cost medium. An alkaline protease producer strain NRC-15 was isolated from Egyptian soil sample. The cultural, morphological, physiological characters and chemotaxonomic evidence strongly indicated that the NRC-15 strain represents a novel species of the genus Streptomyces, hence the name Strptomyces pseudogrisiolus NRC-15. The culture conditions for higher protease production by NRC-15 were optimized with respect to carbon and nitrogen sources, metal ions, pH and temperature. Maximum protease production was obtained in the medium supplemented with 1% glucose, 1% yeast extract, 6% NaCl and 100 μmol/L of Tween 20, initial pH 9.0 at 50 °C for 96 h. The current results confirm that for this strain, a great ability to produce alkaline proteases, which supports the use of applications in industry.

  13. Optimum Production and Characterization of an Acid Protease from Marine Yeast Metschnikowia reukaufii W6b

    Institute of Scientific and Technical Information of China (English)

    LI Jing; PENG Ying; WANG Xianghong; CHI Zhenming

    2010-01-01

    The marine yeast strain W6b isolated from sediment of the South China Sea was found to produce a cell-bound acid protease.The crude acid protease produced by this marine yeast showed the highest activity at pH 3.5 and 40 ℃.The optimal pH and temperature for the crude acid protease were in agreement with those for acid protease produced by the terrestrial yeasts.The optimal medium of the acid protease production was seawater containing 1.0% glucose,1.5% casein,and 0.5% yeast extract,and the optimal cultivation conditions of the acid protease production were pH 4.0,a temperature of 25 ℃ and a shaking speed of 140 rmin-1.Under the optimal conditions,72.5 UmL-1 of acid protease activity could be obtained in cell suspension within 48 h of fermentation at shake flask level.The acid protease production was induced by high-molecular-weight nitrogen sources and repressed by low-molecular-weight nitrogen sources.Skimmed-milk-clotting test showed that the crude acid protease from the cell suspension of the yeast W6b had high skimmed milk coagulability.The acid protease produced by M.reukaufii W6b may have highly potential applications in cheese,food and fermentation industries.

  14. Optimum production and characterization of an acid protease from marine yeast Metschnikowia reukaufii W6b

    Science.gov (United States)

    Li, Jing; Peng, Ying; Wang, Xianghong; Chi, Zhenming

    2010-12-01

    The marine yeast strain W6b isolated from sediment of the South China Sea was found to produce a cell-bound acid protease. The crude acid protease produced by this marine yeast showed the highest activity at pH 3.5 and 40 °C. The optimal pH and temperature for the crude acid protease were in agreement with those for acid protease produced by the terrestrial yeasts. The optimal medium of the acid protease production was seawater containing 1.0% glucose, 1.5% casein, and 0.5% yeast extract, and the optimal cultivation conditions of the acid protease production were pH 4.0, a temperature of 25 °C and a shaking speed of 140 rmin-1. Under the optimal conditions, 72.5 UmL-1 of acid protease activity could be obtained in cell suspension within 48 h of fermentation at shake flask level. The acid protease production was induced by high-molecular-weight nitrogen sources and repressed by low-molecular-weight nitrogen sources. Skimmed-milk-clotting test showed that the crude acid protease from the cell suspension of the yeast W6b had high skimmed milk coagulability. The acid protease produced by M. reukaufii W6b may have highly potential applications in cheese, food and fermentation industries.

  15. Crystallization conditions of porphyritic high-K calc-alkaline granitoids in the extreme northeastern Borborema Province, NE Brazil, and geodynamic implications

    Science.gov (United States)

    Campos, Benedita Cleide Souza; Vilalva, Frederico Castro Jobim; Nascimento, Marcos Antônio Leite do; Galindo, Antônio Carlos

    2016-10-01

    An integrated textural and chemical study on amphibole, biotite, plagioclase, titanite, epidote, and magnetite was conducted in order to estimate crystallization conditions, along with possible geodynamic implications, for six Ediacaran porphyritic high-K calc-alkaline granite plutons (Monte das Gameleiras, Barcelona, Acari, Caraúbas, Tourão, and Catolé do Rocha) intrusive into Archean to Paleoproterozoic rocks of the São José do Campestre (SJCD) and Rio Piranhas-Seridó (RPSD) domains, northern Borborema Province. The studied rocks include mainly porphyritic leucocratic monzogranites, as well as quartz-monzonites and granodiorites. Textures are marked by K-feldspar megacrysts (5-15 cm long) in a fine-to medium-grained matrix composed of quartz, plagioclase, amphibole, biotite, as well as titanite, epidote, Fesbnd Ti oxides, allanite, apatite, and zircon as accessory minerals. Amphibole, biotite and titanite share similar compositional variations defined by increasing Al and Fe, and decreasing Mg contents from the plutons emplaced into the SJCP (Monte das Gameleiras and Barcelona) towards those in the RPSD (Acari, Caraúbas, Tourão, and Catolé do Rocha). Estimated intensive crystallization parameters reveal a weak westward range of increasing depth of emplacement, pressure and temperature in the study area. The SJCD plutons (to the east) crystallized at shallower crustal depths (14-21 km), under slightly lower pressure (3.8-5.5 kbar) and temperature (701-718 °C) intervals, and high to moderate oxygen fugacity conditions (+0.8 plutons (to the west) were emplaced at slightly deeper depths (18-23 km), under higher, yet variable pressures (4.8-6.2 kbar), temperatures (723-776 °C), and moderate to low oxygen fugacity conditions (-1.0 < ΔFQM < +1.8). These results reinforce the contrasts between the tectono-strutuctural domains of São José do Campestre and Rio Piranhas-Seridó in the northern Borborema Province.

  16. Low serum alkaline phosphatase activity in Wilson's disease.

    Science.gov (United States)

    Shaver, W A; Bhatt, H; Combes, B

    1986-01-01

    Low values for serum alkaline phosphatase activity were observed early in the course of two patients with Wilson's disease presenting with the combination of severe liver disease and Coombs' negative acute hemolytic anemia. A review of other cases of Wilson's disease revealed that 11 of 12 patients presenting with hemolytic anemia had values for serum alkaline phosphatase less than their respective sex- and age-adjusted mean values; in eight, serum alkaline phosphatase activity was less than the lower value for the normal range of the test. Low values for serum alkaline phosphatase were much less common in Wilson's disease patients with more chronic forms of presentation. Copper added in high concentration to serum in vitro did not have an important effect on serum alkaline phosphatase activity. The mechanism responsible for the decrease in serum alkaline phosphatase activity in patients is uncertain.

  17. The Place of protease inhibitors in antiretroviral treatment

    Directory of Open Access Journals (Sweden)

    S.B. Tenore

    2009-10-01

    Full Text Available With the introduction of highly active antiretroviral therapy, a number of drugs have been developed. The best choice concerning which antiretroviral analogs to start is always under discussion, especially in the choice between non-nucleoside reverse transcriptase inhibitors-based therapies and ritonavir-boosted protease inhibitors. Both are proven to control viral replication and lead to immunological gain. The choice between a non-nucleoside analog reverse transcriptase inhibitor and a protease inhibitor as a third antiretroviral drug in the therapy should consider factors related to the individual, as well as the inclusion of the best therapy in the patient's daily activities and potential adherence. The protease inhibitor-based therapies showed similar efficacy among the various inhibitors with characteristics concerning the adverse events from each medicine. For the treatment of protease-resistant patients, darunavir and tipranavir showed good efficacy with higher genetic barrier to resistance.

  18. A computational module assembled from different protease family motifs identifies PI PLC from Bacillus cereus as a putative prolyl peptidase with a serine protease scaffold.

    Science.gov (United States)

    Rendón-Ramírez, Adela; Shukla, Manish; Oda, Masataka; Chakraborty, Sandeep; Minda, Renu; Dandekar, Abhaya M; Ásgeirsson, Bjarni; Goñi, Félix M; Rao, Basuthkar J

    2013-01-01

    Proteolytic enzymes have evolved several mechanisms to cleave peptide bonds. These distinct types have been systematically categorized in the MEROPS database. While a BLAST search on these proteases identifies homologous proteins, sequence alignment methods often fail to identify relationships arising from convergent evolution, exon shuffling, and modular reuse of catalytic units. We have previously established a computational method to detect functions in proteins based on the spatial and electrostatic properties of the catalytic residues (CLASP). CLASP identified a promiscuous serine protease scaffold in alkaline phosphatases (AP) and a scaffold recognizing a β-lactam (imipenem) in a cold-active Vibrio AP. Subsequently, we defined a methodology to quantify promiscuous activities in a wide range of proteins. Here, we assemble a module which encapsulates the multifarious motifs used by protease families listed in the MEROPS database. Since APs and proteases are an integral component of outer membrane vesicles (OMV), we sought to query other OMV proteins, like phospholipase C (PLC), using this search module. Our analysis indicated that phosphoinositide-specific PLC from Bacillus cereus is a serine protease. This was validated by protease assays, mass spectrometry and by inhibition of the native phospholipase activity of PI-PLC by the well-known serine protease inhibitor AEBSF (IC50 = 0.018 mM). Edman degradation analysis linked the specificity of the protease activity to a proline in the amino terminal, suggesting that the PI-PLC is a prolyl peptidase. Thus, we propose a computational method of extending protein families based on the spatial and electrostatic congruence of active site residues.

  19. A computational module assembled from different protease family motifs identifies PI PLC from Bacillus cereus as a putative prolyl peptidase with a serine protease scaffold.

    Directory of Open Access Journals (Sweden)

    Adela Rendón-Ramírez

    Full Text Available Proteolytic enzymes have evolved several mechanisms to cleave peptide bonds. These distinct types have been systematically categorized in the MEROPS database. While a BLAST search on these proteases identifies homologous proteins, sequence alignment methods often fail to identify relationships arising from convergent evolution, exon shuffling, and modular reuse of catalytic units. We have previously established a computational method to detect functions in proteins based on the spatial and electrostatic properties of the catalytic residues (CLASP. CLASP identified a promiscuous serine protease scaffold in alkaline phosphatases (AP and a scaffold recognizing a β-lactam (imipenem in a cold-active Vibrio AP. Subsequently, we defined a methodology to quantify promiscuous activities in a wide range of proteins. Here, we assemble a module which encapsulates the multifarious motifs used by protease families listed in the MEROPS database. Since APs and proteases are an integral component of outer membrane vesicles (OMV, we sought to query other OMV proteins, like phospholipase C (PLC, using this search module. Our analysis indicated that phosphoinositide-specific PLC from Bacillus cereus is a serine protease. This was validated by protease assays, mass spectrometry and by inhibition of the native phospholipase activity of PI-PLC by the well-known serine protease inhibitor AEBSF (IC50 = 0.018 mM. Edman degradation analysis linked the specificity of the protease activity to a proline in the amino terminal, suggesting that the PI-PLC is a prolyl peptidase. Thus, we propose a computational method of extending protein families based on the spatial and electrostatic congruence of active site residues.

  20. Some durability aspects of hybrid alkaline cements

    Directory of Open Access Journals (Sweden)

    Donatello S.

    2014-04-01

    Full Text Available Blended cements that contain a high content of fly ash and a low content of Portland cement typically suffer from low early strength development and long setting times. Recently, one method of overcoming these problems has been to use an alkali activator to enhance the reactivity of fly ash particles at early ages. Such cements can be grouped under the generic term “hybrid alkaline cements”, where both cement clinker and fly ash, encouraged by the presence of alkalis, are expected to contribute to cementitious gel formation. The work presented here examines some of the durability aspects of high fly ash content hybrid alkaline cement. Specifically, the aspects investigated were: exposure at high temperatures (up to 1000°C, resistance to immersion in aggressive solutions and susceptibility to the alkali aggregate reaction. All tests were repeated with a commercially available sulfate resistant Portland cement for comparison. When exposed to high temperatures, the hybrid alkaline cement showed strikingly different behaviour compared to the control Portland cement, showing fewer micro-cracks and maintaining residual compressive strengths at least equal to original strengths. Beyond 700°C, the hybrid alkaline cement began to sinter, which resulted in shrinkage of around 5% and a 100% increase in residual compressive strengths. No such sintering event was noted in the control Portland cement, which showed a drastic loss in residual compressive strengths upon heating. In immersion tests, the hybrid alkaline cement possessed excellent resistance to sulfate and seawater attack, similar to the control sulfate resistant cement. Both cements were however severely degraded by immersion in 0.1M HCl for 90 days. Both binders complied with the accelerated alkali-aggregate test but when this test was extended, the hybrid alkaline binder showed much greater dimensional stability. Possible reasons for the differences in durability behaviour in both cements

  1. Novel peptide-based protease inhibitors

    DEFF Research Database (Denmark)

    Roodbeen, Renée

    This thesis describes the design and synthesis of peptide-based serine protease inhibitors. The targeted protease, urokinase-type plasminogen activator (uPA) activates plasminogen, which plays a major role in cancer metastasis. The peptide upain-2 (S 1 ,S 12-cyclo-AcCSWRGLENHAAC-NH2) is a highly......, the disulfide bridge was replaced with amide bonds of various lengths. The novel peptides did not retain their inhibitory activity, but formed the basis for another strategy. Second, bicyclic peptides were obtained by creating head-to-tail cyclized peptides that were made bicyclic by the addition of a covalent...... increased. Finally, the effect of multivalent display of upain-2 was investigated. Several dimers of upain-2 were made and the attachment of upain-2 via the Copper-catalyzed Azide-Alkyne Cycloaddition (CuAAC) onto an alkyne functionalized carbohydrate scaffold was investigated. Besides the synthesis...

  2. Feces Derived Allergens of Tyrophagus putrescentiae Reared on Dried Dog Food and Evidence of the Strong Nutritional Interaction between the Mite and Bacillus cereus Producing Protease Bacillolysins and Exo-chitinases.

    Science.gov (United States)

    Erban, Tomas; Rybanska, Dagmar; Harant, Karel; Hortova, Bronislava; Hubert, Jan

    2016-01-01

    Tyrophagus putrescentiae (Schrank, 1781) is an emerging source of allergens in stored products and homes. Feces proteases are the major allergens of astigmatid mites (Acari: Acaridida). In addition, the mites are carriers of microorganisms and microbial adjuvant compounds that stimulate innate signaling pathways. We sought to analyze the mite feces proteome, proteolytic activities, and mite-bacterial interaction in dry dog food (DDF). Proteomic methods comprising enzymatic and zymographic analysis of proteases and 2D-E-MS/MS were performed. The highest protease activity was assigned to trypsin-like proteases; lower activity was assigned to chymotrypsin-like proteases, and the cysteine protease cathepsin B-like had very low activity. The 2D-E-MS/MS proteomic analysis identified mite trypsin allergen Tyr p3, fatty acid-binding protein Tyr p13 and putative mite allergens ferritin (Grp 30) and (poly)ubiquitins. Tyr p3 was detected at different positions of the 2D-E. It indicates presence of zymogen at basic pI, and mature-enzyme form and enzyme fragment at acidic pI. Bacillolysins (neutral and alkaline proteases) of Bacillus cereus symbiont can contribute to the protease activity of the mite extract. The bacterial exo-chitinases likely contribute to degradation of mite exuviae, mite bodies or food boluses consisting of chitin, including the peritrophic membrane. Thus, the chitinases disrupt the feces and facilitate release of the allergens. B. cereus was isolated and identified based on amplification and sequencing of 16S rRNA and motB genes. B. cereus was added into high-fat, high-protein (DDF) and low-fat, low-protein (flour) diets to 1 and 5% (w/w), and the diets palatability was evaluated in 21-day population growth test. The supplementation of diet with B. cereus significantly suppressed population growth and the suppressive effect was higher in the high-fat, high-protein diet than in the low-fat, low-protein food. Thus, B. cereus has to coexist with the mite in

  3. Feces derived allergens of Tyrophagus putrescentiae reared on dried dog food and evidence of the strong nutritional interaction between the mite and Bacillus cereus producing protease bacillolysins and exo-chitinases

    Directory of Open Access Journals (Sweden)

    Tomas eErban

    2016-02-01

    Full Text Available Tyrophagus putrescentiae (Schrank, 1781 is an emerging source of allergens in stored products and homes. Feces proteases are the major allergens of astigmatid mites (Acari: Acaridida. In addition, the mites are carriers of microorganisms and microbial adjuvant compounds that stimulate innate signaling pathways. We sought to analyze the mite feces proteome, proteolytic activities and mite-bacterial interaction in dry dog food. Proteomic methods comprising enzymatic and zymographic analysis of proteases and 2D-E-MS/MS were performed. The highest protease activity was assigned to trypsin-like proteases; lower activity was assigned to chymotrypsin-like proteases, and the cysteine protease cathepsin B-like had very low activity. The 2D-E-MS/MS proteomic analysis identified mite trypsin allergen Tyr p3, fatty acid-binding protein Tyr p13 and putative mite allergens ferritin (Grp 30 and (polyubiquitins. Tyr p3 was detected at different positions of the 2D-E. It indicates presence of zymogen at basic pI, and mature-enzyme form and enzyme fragment at acidic pI. Bacillolysins (neutral and alkaline proteases of Bacillus cereus symbiont can contribute to the protease activity of the mite extract. The bacterial exo-chitinases likely contribute to degradation of mite exuviae, mite bodies or food boluses consisting of chitin, including the peritrophic membrane. Thus, the chitinases disrupt the feces and facilitate release of the allergens. B. cereus was isolated and identified based on amplification and sequencing of 16S rRNA and motB genes. B. cereus was added into high-fat, high-protein (dry dog food and low-fat, low-protein (flour diets to 1% and 5% (w/w, and the diets palatability was evaluated in 21-day population growth test. The supplementation of diet with B. cereus significantly suppressed population growth and the suppressive effect was higher in the high-fat, high-protein diet than in the low-fat, low-protein food. Thus, B. cereus has to coexist

  4. Recent Alkaline Lakes: Clues to Understanding the Evolution of Early Planetary Alkaline Oceans and Biogenesis

    Science.gov (United States)

    Kempe, S.; Hartmann, J.; Kazmierczak, J.

    2008-09-01

    Abstract New models suggest that terrestrial weathering consumes 0.26GtC/a (72% silicate-, 28% carbonateweathering), equivalent to a loss of one atmospheric C content every 3700a. Rapid weathering leads in volcanic areas to alkaline conditions, illustrated by the crater lake of Niuafo`ou/Tonga and Lake Van/Turkey, the largest soda lake on Earth. Alkaline conditions cause high CaCO3 supersaturation, permineralization of algal mats and growth of stromatolites. Alkaline conditions can nearly depress free [Ca2+] to levels necessary for proteins to function. Therefore early oceans on Earth (and possibly on Mars) should have been alkaline (i.e. "Soda Oceans"). Recent findings of MgSO4 in top soils on Mars may be misleading about the early history of martian oceans.

  5. Application of a cell-based protease assay for testing inhibitors of picornavirus 3C proteases

    NARCIS (Netherlands)

    van der Linden, Lonneke; Ulferts, Rachel; Nabuurs, Sander B; Kusov, Yuri; Liu, Hong; George, Shyla; Lacroix, Céline; Goris, Nesya; Lefebvre, David; Lanke, Kjerstin H W; De Clercq, Kris; Hilgenfeld, Rolf; Neyts, Johan; van Kuppeveld, Frank J M

    2014-01-01

    Proteolytical cleavage of the picornaviral polyprotein is essential for viral replication. Therefore, viral proteases are attractive targets for anti-viral therapy. Most assays available for testing proteolytical activity of proteases are performed in vitro, using heterologously expressed proteases

  6. Use of the Protease Fluorescent Detection Kit to Determine Protease Activity

    OpenAIRE

    Cupp-Enyard, Carrie

    2009-01-01

    The Protease Fluorescent Detection Kit provides ready-to-use reagents for detecting the presence of protease activity. This simple assay to detect protease activity uses casein labeled with fluorescein isothiocyanate (FITC) as the substrate.

  7. Serine proteases, serine protease inhibitors, and protease-activated receptors: roles in synaptic function and behavior.

    Science.gov (United States)

    Almonte, Antoine G; Sweatt, J David

    2011-08-17

    Serine proteases, serine protease inhibitors, and protease-activated receptors have been intensively investigated in the periphery and their roles in a wide range of processes-coagulation, inflammation, and digestion, for example-have been well characterized (see Coughlin, 2000; Macfarlane et al., 2001; Molinari et al., 2003; Wang et al., 2008; Di Cera, 2009 for reviews). A growing number of studies demonstrate that these protein systems are widely expressed in many cell types and regions in mammalian brains. Accumulating lines of evidence suggest that the brain has co-opted the activities of these interesting proteins to regulate various processes underlying synaptic activity and behavior. In this review, we discuss emerging roles for serine proteases in the regulation of mechanisms underlying synaptic plasticity and memory formation. Copyright © 2011 Elsevier B.V. All rights reserved.

  8. Post-prandial protease activity in the digestive tract of African catfish, Clarias gariepinus, larvae fed decapsulated cysts of Artemia

    NARCIS (Netherlands)

    García Ortega, A.; Verreth, J.; Segner, H.

    2000-01-01

    The alkaline proteolytic activity in the gut of African catfish larvae was studied during short time ranges from 30 min to 4 h after ingestion of decapsulated Artemia cysts. The variation in total protease and trypsin activities during the day was monitored during starvation, after one single meal i

  9. Performance and Application of Corrosion Inhibitor TH-658B in High Hardness and High Alkalinity Water%新型高硬高碱水缓蚀阻垢剂TH-658B的应用性能

    Institute of Scientific and Technical Information of China (English)

    贺茂才; 齐晓婧; 高灿柱

    2012-01-01

    针对华北、西北等地补充水硬度、碱度高,浓缩后极易引起结垢和腐蚀的难题,开发了高硬、高碱水缓蚀阻垢剂TH-658B。采用静态阻碳酸钙垢试验、阻硫酸钙垢试验、梯度浓缩试验等研究了TH658B的性能。结果表明,相同的药剂浓度,TH-658B的碳酸钙阻垢率均高于ATMP和HEDP且TH-658B的碳酸钙和硫酸钙阻垢率均符合Q/SY126—2007要求;TH-658B可以使高硬、高碱水浓缩至钙硬总碱之和大于1500mg/L而不会导致系统结垢。此外,参照GB/T18175-2000,在TH-658B的存在下,高硬、高碱配水(〉1500mg/L)中的碳钢、不锈钢和铜的腐蚀速率分别为0.036、0.0018、0.0012mm/a,均符合GB50050—2007的要求。通过华北一电厂的现场应用,证明了TH-658B对高硬、高碱,特别是钙硬总碱之和达到1500mg/L以上的高浓缩倍率循环水系统具有良好的缓蚀和阻垢效果。%Aiming at the scaling and corrosion problem of high hardness and alkalinity water in north and northwest of China, a new type of scale and corrosion inhibitor TH-658B was developed. The performance of TH-658B was evaluated by means of scale inhibition test, gradient concentration test and rotary coupon corrosion test. The results show that the calcium carbonate scale inhibition rate of TH-658B was higher than ATMP and HEDP under the same concentration condition. The calcium carbonate and calcium sulfate scale inhibition rate of TH-658B can meet the standard requirements of Q/SY 126--2007. Gradient enrichment test results indicate that the summation of calcium hardness and total alkalinity could reach more than 1500 mg/L as TH-658B existed in water, which is much higher than the standard requirements of GBS0050- 2007. Furthermore, TH-658B has good inhibition corrosion performance for carbon steel, stainless steel and copper in high hardness and alkalinity water. The corrosion rates for carbon steel, stainless steel and

  10. Production and Characterization of Keratinolytic Protease from New Wool-Degrading Bacillus Species Isolated from Egyptian Ecosystem

    Directory of Open Access Journals (Sweden)

    Mohamed A. Hassan

    2013-01-01

    Full Text Available Novel keratin-degrading bacteria were isolated from sand soil samples collected from Minia Governorate, Egypt. In this study, the isolates were identified as Bacillus amyloliquefaciens MA20 and Bacillus subtilis MA21 based on morphological and biochemical characteristics as well as 16S rRNA gene sequencing. B. amyloliquefaciens MA20 and B. subtilis MA21 produced alkaline keratinolytic serine protease when cultivated in mineral medium containing 1% of wool straight off sheep as sole carbon and nitrogen source. The two strains were observed to degrade wool completely to powder at pH 7 and 37°C within 5 days. Under these conditions the maximum activity of proteases produced by B. amyloliquefaciens MA20 and B. subtilis MA21 was 922 and 814 U/ml, respectively. The proteases exhibited optimum temperature and pH at 60°C and 9, respectively. However, the keratinolytic proteases were stable in broad range of temperature and pH values towards casein Hammerstein. Furthermore the protease inhibitor studies indicated that the produced proteases belong to serine protease because of their sensitivity to PMSF while they were inhibited partially in presence of EDTA. The two proteases are stable in most of the used organic solvents and enhanced by metals suggesting their potential use in biotechnological applications such as wool industry.

  11. Production and Characterization of Keratinolytic Protease from New Wool-Degrading Bacillus Species Isolated from Egyptian Ecosystem

    Science.gov (United States)

    Hassan, Mohamed A.; Haroun, Bakry M.; Amara, Amro A.; Serour, Ehab A.

    2013-01-01

    Novel keratin-degrading bacteria were isolated from sand soil samples collected from Minia Governorate, Egypt. In this study, the isolates were identified as Bacillus amyloliquefaciens MA20 and Bacillus subtilis MA21 based on morphological and biochemical characteristics as well as 16S rRNA gene sequencing. B. amyloliquefaciens MA20 and B. subtilis MA21 produced alkaline keratinolytic serine protease when cultivated in mineral medium containing 1% of wool straight off sheep as sole carbon and nitrogen source. The two strains were observed to degrade wool completely to powder at pH 7 and 37°C within 5 days. Under these conditions the maximum activity of proteases produced by B. amyloliquefaciens MA20 and B. subtilis MA21 was 922 and 814 U/ml, respectively. The proteases exhibited optimum temperature and pH at 60°C and 9, respectively. However, the keratinolytic proteases were stable in broad range of temperature and pH values towards casein Hammerstein. Furthermore the protease inhibitor studies indicated that the produced proteases belong to serine protease because of their sensitivity to PMSF while they were inhibited partially in presence of EDTA. The two proteases are stable in most of the used organic solvents and enhanced by metals suggesting their potential use in biotechnological applications such as wool industry. PMID:23936776

  12. Production, partial purification and characterization of protease from a phytopathogenic fungi Alternaria solani (Ell. and Mart.) Sorauer.

    Science.gov (United States)

    Chandrasekaran, Murugesan; Sathiyabama, Muthukrishnan

    2014-08-01

    An alkaline serine protease producing strain Alternaria solani was optimized for its enzyme production under submerged conditions. The maximum production of protease by A. solani was achieved by using sodium nitrate at the optimum concentration of 0.2% w/v. A. solani produced higher quantities (3.75 [unit/mg of protein]) of an inducible extracellular proteases on day 9 after incubation in czapek's dox broth medium amended with 1% casein as an inducer at pH 8.5, temperature 27 °C and 3% sucrose as carbon source. Extracellular proteases were precipitated by ammonium sulphate saturation (80%) method and purified on Sephadex G-100 column chromatography. The molecular mass of SDS-PAGE and Sephadex G-100 Column Gel permeation chromatography purified protease was estimated to 42 kDa. In addition, trypsin digestion of 42 kDa protein band was carried out and analyzed by MALDI-TOF for the identification of protease. The sequence IKELATNGVVTNVK (378-391) segment of the alkaline serine protease was found by using MS/MS spectrum at 1485 m/z from the purified fraction. It showed optimal activity at 50 °C and pH 9-10 and broad pH stability between pH 6-12. The protease activity was inhibited by phenyl methyl sulfonyl fluoride (PMSF), all the results indicated that the presence of a serine residue in the active site and is thus most likely a member of the serine protease family. This may function as a virulence protein during pathogenesis by A. solani. The results suggested that the presence of appreciable extracellular proteolytic activity in filamentous fungi may serve as a marker of their phytopathogenicity. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Metal-based antimicrobial protease inhibitors.

    Science.gov (United States)

    Kellett, A; Prisecaru, A; Slator, C; Molphy, Z; McCann, M

    2013-01-01

    Limitations associated with the production cost, metabolic instability, side-effects, resistance and poor pharmacokinetics of organic protease inhibitors (PIs), which form an essential component of the front line HAART treatment for HIV, have fuelled efforts into finding novel, transition metal-based alternatives. Some of the attractive features of metalbased therapeutics include synthetic simplicity, solubility control, redox capability, expansion of coordination number and topography matching of the complex to the protein's active site. Building asymmetry into the complex, which may offer better discrimination between host and rogue cell, can readily be achieved through coordination of chiral ligands to the metal centre. Although the scope of this review has been limited to metal-based agents that have been reported to bind/inhibit HIV-1 and parasitic proteases, some desirables, such as high activity, low dosage, minimal toxicity, crossinhibition, unique binding modes and selectivity, have already been delivered. The variability of the d-block metals, coupled with the availability of designer organic ligands, augers well for the future development of clinical metallo-drugs for deployment against protease-associated, fatal diseases.

  14. Direct visualization of protease action on collagen triple helical structure.

    Directory of Open Access Journals (Sweden)

    Gabriel Rosenblum

    Full Text Available Enzymatic processing of extracellular matrix (ECM macromolecules by matrix metalloproteases (MMPs is crucial in mediating physiological and pathological cell processes. However, the molecular mechanisms leading to effective physiological enzyme-ECM interactions remain elusive. Only scant information is available on the mode by which matrix proteases degrade ECM substrates. An example is the enzymatic degradation of triple helical collagen II fragments, generated by the collagenase MMP-8 cleavage, during the course of acute inflammatory conditions by gelatinase B/MMP-9. As is the case for many other matrix proteases, it is not clear how MMP-9 recognizes, binds and digests collagen in this important physiological process. We used single molecule imaging to directly visualize this protease during its interaction with collagen fragments. We show that the initial binding is mediated by the diffusion of the protease along the ordered helix on the collagen (3/4 fragment, with preferential binding of the collagen tail. As the reaction progressed and prior to collagen degradation, gelatin-like morphologies resulting from the denaturation of the triple helical collagen were observed. Remarkably, this activity was independent of enzyme proteolysis and was accompanied by significant conformational changes of the working protease. Here we provide the first direct visualization of highly complex mechanisms of macromolecular interactions governing the enzymatic processing of ECM substrates by physiological protease.

  15. Isolation and characterization of protease from Bacillus subtilis 1012M15

    Directory of Open Access Journals (Sweden)

    ELFI SUSANTI

    2003-01-01

    Full Text Available A local strain of Bacillus sp. BAC4, is known to produce penicillin G acylase (PGA enzyme with relatively high activity. This strain secretes the PGA into the culture medium. However, it has been reported that PGA activity fall and rise during culture, and the activity plummets during storege at –200C, which probably due to usage protease activity of Bacillus sp. BAC4. To study the possible use of Bacillus subtilis 1012M15 as a host cell for cloning the pga gene from Bacillus sp. BAC4, the protease activity of Bacillus subtilis 1012M15 were studied. Protease activity was determined by Horikoshi method. In this experiment, maximum protease activity in Bacillus subtilis 1012M15 culture was obsereved after 8 hours. At this optimum condition, protease activity of Bacillus sp. BAC4 is five time higher than that of Bacillus subtilis 1012M15. This situation promised the possible usage of Bacillus subtilis 1012M15 as a host cell for pga expression. For protease characterization, the bacterial culture had been separated from the cell debris by centrifugation. The filtrate was concentrated by freeze drying, fractionated by ammonium sulphate, dialyzed in selovan tube, and then fractionated by ion exchance chromatography employing DEAE-cellulose. The five peaks resulted indicated the presence of five protease. Based on inhibitor and activator influence analysis, it could be concluded that proteases from Bacillus subtilis 1012M15 contained of serin protease as well as metalloprotease and serin protease mixture.

  16. Screening of protease-producing marine yeasts for production of the bioactive peptides

    Institute of Scientific and Technical Information of China (English)

    NI Xiumei; CHI Zhenming; LIU Zhiqiang; YUE Lixi

    2008-01-01

    Over 400 yeast strains from seawater and sediments were obtained,but only five strains named HN2-3,N13d,N13C,Mb5 and HN3-2 among them could form clear zones around their colonies on the double plates with 2.0% casein.Peptides in the hydroly-sate produced by the proteases from strains HN2-3 and N13d had higher angiotensin Ⅰ-converting-enzyme (ACE)-inhibitory ac-tivity.The two marine yeast strains were identified to be Aureobasidium pullulans according to the results of routine yeast identifi-cation and molecular methods.After purification of the proteases from the two marine yeast strains,it was found that the optimal pH for them was both 9.0,both of them were serine alkaline protease.However,the optimal temperature for the protease from the strain HN2-3 was 52℃ while that from strain N13d was 48℃.ACE-inhibitory activity of the peptides in the hydrolysate of shrimp protein produced by the purified protease from the strain HN2-3 was the highest while antioxidant activity in the hydroly-sate of spirulina protein produced by the purified protease from the strain N13d was the highest.

  17. Diode Pumped Alkaline Laser System: A High Powered, Low SWaP Directed Energy Option for Ballistic Missile Defense High-Level Summary - April 2017

    Energy Technology Data Exchange (ETDEWEB)

    Wisoff, P. J. [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)

    2017-04-28

    The Diode-Pumped Alkali Laser (DPAL) system is an R&D effort funded by the Missile Defense Agency (MDA) underway at Lawrence Livermore National Laboratory (LLNL). MDA has described the characteristics needed for a Boost Phase directed energy (DE) weapon to work against ICBM-class threat missiles. In terms of the platform, the mission will require a high altitude Unmanned Aerial Vehicle (UAV) that can fly in the “quiet” stratosphere and display long endurance – i.e., days on station. In terms of the laser, MDA needs a high power, low size and weight laser that could be carried by such a platform and deliver lethal energy to an ICBM-class threat missile from hundreds of kilometers away. While both the military and industry are pursuing Directed Energy for tactical applications, MDA’s objectives pose a significantly greater challenge than other current efforts in terms of the power needed from the laser, the low size and weight required, and the range, speed, and size of the threat missiles. To that end, MDA is funding two R&D efforts to assess the feasibility of a high power (MWclass) and low SWaP (size, weight and power) laser: a fiber combining laser (FCL) project at MIT’s Lincoln Laboratory, and LLNL’s Diode-Pumped Alkali Laser (DPAL) system.

  18. Improving volatile fatty acids production by exploiting the residual substrates in post-fermented sludge: Protease catalysis of refractory protein.

    Science.gov (United States)

    Yin, Bo; Liu, Hongbo; Wang, Yuanyuan; Bai, Jie; Liu, He; Fu, Bo

    2016-03-01

    The real cause to the low yield of volatile fatty acids (VFAs), from inhibition or low biodegradation, is uncertain in sludge anaerobic fermentation. In this study, poor biodegradability of proteins and fast decrease of the indigenous hydrolase activity in the residual post-fermented sludge were found to be the major reasons. With the addition of trypsin or alkaline protease in residual post-fermented sludge after primary alkaline fermentation, degradation efficiency of refractory protein increased by 33.6% and 34.8%, respectively. Accordingly, the VFAs yields were improved by 69.7% and 106.1%, respectively. Furthermore, the activities of added trypsin and alkaline protease could maintain at 13.52 U/mL and 19.11 U/mL in the alkaline fermentation process. This study demonstrated that exploiting the refractory proteins in residual post-fermented sludge by protease addition seems to be a very promising way for improving VFAs yield of conventional alkaline fermentations with waste activated sludge. Copyright © 2015 Elsevier Ltd. All rights reserved.

  19. Analysis of HIV- type 1 protease and reverse transcriptase in Brazilian children failing highly active antiretroviral therapy (HAART Análise da protease e transcriptase reversa do HIV-1 em crianças com falha terapêutica em uso de terapia anti-retroviral altamente eficaz (HAART

    Directory of Open Access Journals (Sweden)

    Daisy Maria Machado

    2005-02-01

    Full Text Available The aim of this study was to evaluate the genotypic resistance profiles of HIV-1 in children failing highly active antiretroviral therapy (HAART. Forty-one children (median age = 67 months receiving HAART were submitted to genotypic testing when virological failure was detected. cDNA was extracted from PBMCs and amplified by nested PCR for the reverse transcriptase and protease regions of the pol gene. Drug resistance genotypes were determined from DNA sequencing. According to the genotypic analysis, 12/36 (33.3% and 6/36 (16.6% children showed resistance and possible resistance, respectively, to ZDV; 5/36 (14% and 4/36 (11.1%, respectively, showed resistance and possible resistance to ddI; 4/36 (11.1% showed resistance to 3TC and D4T; and 3/36 (8.3% showed resistance to Abacavir. A high percentage (54% of children exhibited mutations conferring resistance to NNRTI class drugs. Respective rates of resistance and possible resistance to PIs were: RTV (12.2%, 7.3%; APV (2.4%, 12.1%; SQV(0%, 12.1%; IDV (14.6%, 4.9%, NFV (22%, 4.9%, LPV/RTV (2.4%, 12.1%. Overall, 37/41 (90% children exhibited virus with mutations related to drug resistance, while 9% exhibited resistance to all three antiretroviral drug classes.O objetivo deste estudo foi avaliar o perfil de resistência genotípica do HIV-1 em crianças com falha terapêutica ao tratamento anti-retroviral (HAART. Quarenta e uma crianças (idade mediana = 67 meses em uso de HAART foram submetidas ao teste de genotipagem no momento da detecção de falha ao tratamento. Foi realizada extração de cDNA de células periféricas mononucleares e amplificação do mesmo (regiões da transcriptase reversa e protease do gene pol através de PCR-nested. O perfil genotípico foi determinado através do seqüenciamnto de nucleotídeos. De acordo com a análise genotípica, 12/36 (33,3% e 6/36 (16,6% crianças apresentaram, respectivamente, resistência e possível resistência ao AZT; 5/36 (14% e 4/36 (11

  20. Alkaline hydrothermal zeolites synthesized from high SiO{sub 2} and Al{sub 2}O{sub 3} co-disposal fly ash filtrates

    Energy Technology Data Exchange (ETDEWEB)

    Vernon S. Somerset; Leslie F. Petrik; Richard A. White; Michael J. Klink; David Key; Emmanuel I. Iwuoha

    2005-12-01

    A co-disposal reaction was used wherein fly ash (FA) was reacted with acid mine drainage (AMD), to collect filtrates for zeolite synthesis. Raw fly ash as well as fly ash leached with HCl were subjected to the same alkaline hydrothermal zeolite synthesis conditions, as for the co-disposal filtrates, in order to evaluate the zeolitic material obtained. The Si and Al contents of the fly ash (FA) filtrates were used as precursor species for the alkaline hydrothermal conversion of the fly ash filtrates into zeolites. These filtrates were then analysed by XRF spectrometry for quantitative determination of SiO{sub 2} and Al{sub 2}O{sub 3}. The (SiO{sub 2})/(Al{sub 2}O{sub 3}) ratio obtained in the filtrates range from 1.4 to 2.5. The (SiO{sub 2})/(Al{sub 2}O{sub 3}) ratio was used to predict whether the fly ash filtrates could successfully be converted into faujasite zeolitic material by the adopted synthesis procedures. If the (SiO{sub 2})/(Al{sub 2}O{sub 3}) ratio is higher than 1.5 in the co-disposal filtrates, it favours the formation of faujasite. The zeolite synthesis included an alkaline fusion of the co-disposal filtrates, followed by aging for 8 hours and hydrothermal conversion by crystallisation at 100{sup o}C. Different variables were investigated during the synthesis of zeolite to ascertain their influence on the end product. These variables include adding different amounts of deionised water to the FA-related starting material, using different compositions of FA related starting material and different FA:NaOH ratios in fusing the starting material. 15 refs., 3 figs., 2 tabs.

  1. Cenozoic high-K alkaline magmatism and associated Cu-Mo-Au mineralization in the Jinping-Fan Si Pan region, southeastern Ailao Shan-Red River shear zone, southwestern China-northwestern Vietnam

    Science.gov (United States)

    Tran, My Dung; Liu, Junlai; Nguyen, Quang Luat; Chen, Yue; Tang, Yuan; Song, Zhijie; Zhang, Zhaochong; Zhao, Zhidan

    2014-01-01

    The Jinping-Fan Si Pan (JFP) Cenozoic magmatic and Cu-Mo-Au metallogenic belt in the southeastern part of the Ailao Shan shear zone host the Tongchang, Chang‧an, Habo, and Chinh Sang Cu-Mo-Au deposits. These deposits form an integrated epithermal-porphyry regional mineralization system associated with 40-32 Ma high-K alkaline magmatism. The magmatic rocks in the belt have relatively low TiO2 (<0.73 wt%), P2O5 (<0.29 wt%), and FeO* (<4.99 wt%), and high Na2O (2.86-4.75 wt%) and K2O (4.01-7.98 wt%). They also have high contents of incompatible trace elements, and are enriched in LILE (Rb, Ba, K, Sr) and LREE. They have marked Nb, Ta, Ti and P depletion in primitive mantle-normalized spidergrams, and plot close to the EMII mantle field in the Sr-Nd isotopic diagram. These characteristics are similar to those of the Eocene high-K alkaline rocks along the northern Ailao Shan belt, eastern Tibet plateau. The sulfur and lead isotope analyses of sulfide minerals from both the ores and related magmatic rocks confirm the involvement of a magmatic ore fluid. The Cenozoic alkaline intrusions and Cu-Mo-Au mineralization in the JFP were formed prior to the initiation of left-lateral shearing along the Ailao Shan shear zone. The magmas appear to have been derived from enriched mantle, possibly with mixing of materials from the buried Tethyan oceanic lithosphere, and/or crust.

  2. A novel neutral protease from thermophilic Bacillus strain HUTBS62

    Directory of Open Access Journals (Sweden)

    HAZEM AQEL

    2012-01-01

    Full Text Available A novel neutral highly thermostable protease was detected in the culture medium of thermophilic Bacillus strain HUTBS62 isolated from hot-spring located near to the Dead Sea, Jordan. The enzyme was purified by precipitation with 55-60% ammonium sulfate, gel filtration on Sephadex G-100 and DEAE ion exchange chromatography. The enzyme was purified 53-fold with 2% yield. The optimum pH and temperature for catalytic activity of protease was pH 6.8 and 80ºC, respectively, and 31% activity of protease remained even after heat treatment at 100ºC for 60 min. The relative activity of the enzyme was highly stable (90% at 50ºC for 2 h. The half-life of the enzyme at 90ºC, 80ºC and 70ºC was estimated to be 3, 4 and 6 h, respectively. The activation energy of denaturation of purified enzyme was 21.7 kJmol-1. Iron, sodium, calcium, and manganese increased protease activity. On the other hand, magnesium, cobalt and zinc variably decreased the residual activity. But cadmium and copper drastically inhibited the enzyme activity. The enzymatic activity was highly stable in the presence of 1 and 2 mM EDTA at pH 6.8 and 80ºC. The neutral protease therefore could be defined as a highly thermostable with new properties make the present enzyme applicable for many biotechnological purposes.

  3. Effects of Combined High Pressure and Thermal Treatment on Protease Activities in Beef Muscle%高压与加热协同处理对牛肌肉中蛋白酶活性的影响

    Institute of Scientific and Technical Information of China (English)

    马汉军; 周光宏; 余小领; 高海燕; 潘润淑; D. A. Ledward

    2011-01-01

    The effects of combined high pressure (0.1~600 MPa) and temperature (20~60℃) treatment for 20 min on beef protease activities were studied. The results showed that the protease activities decreased with increasing pressure up to 400 MPa at room temperature,after which there was no significant change of them. The content of free amino acid increased significantly at 200 MPa and then the increased speed decreased with the pressure increasing. 400 MPa or higher pressure treatment at room temperature resulted in the increase of tryptic digestibility of protein in beef extract. There was no significant effect on protease activities and free amino acid content in beef after pressurized at 200 MPa,40 ℃ ,but the protease was almost completely inactivated at pH 7. 5 and free amino acid content reduced rapidly after treated at 600 MPa,40 ℃. Part of protease activities survived in beef muscle when heated at 60 ℃. They did not change significantly when treated at 200 MPa,60 ℃ ,but the free amino acid content increased obviously. Treatment of 600 MPa at 60 ℃ resulted in the inactivation of almost all the proteases and the free amino acid content also decreased markedly. The similar changes of tryptic digestibility of protein in beef extract were found when pressure applied at 40~ 60 ℃. 200 MPa treatment did not cause significant changes and 600 MPa treatment induced the tryptic digestibility of protein marked increase. These results suggested that high pressure treatment combined with heating could regulate proteases activities of beef and improve its quality.%在不同温度(20~60℃)和压力(0.1~600 MPa)下处理20 min,对牛肌肉中蛋白酶活性的影响进行了研究.结果显示:室温下,随着处理压力的增加,酶的活力显著下降,而压力达400 MPa及以上时,酶的活力则没有明显变化,同时在pH值为3和7.5时酶的活性几乎完全丧失.200 MPa以下的压力处理使肌肉中游离氨基酸总量显著上升,压力

  4. Protease activity in gut of Daphnia magna: evidence for trypsin and chymotrypsin enzymes.

    Science.gov (United States)

    von Elert, Eric; Agrawal, Manish Kumar; Gebauer, Christine; Jaensch, Heike; Bauer, Ulrike; Zitt, Anja

    2004-03-01

    Two major protease activities were present in gut homogenates of the cladoceran crustacean Daphnia magna: (i) a trypsin activity that hydrolysed the synthetic substrate N-benzoyl-dl-arginine p-nitroanilide and was strongly inhibited by N-p-tosyl-lysine chloroketone (TLCK) and 4-(amidinophenyl)methanesulfonyl fluoride (APMSF) and not inhibited by chymostatin; and (ii) a chymotrypsin activity that hydrolysed synthetic chymotrypsin substrates containing more than one amino acid, did not hydrolyse N-benzoyl-l-tyrosine p-nitroanilide, and was strongly inhibited by chymostatin and not by TLCK and APMSF. Both activities had alkaline pH optima (pH 7-10), but were shown to be due to distinct types of proteases. These two enzyme activities accounted for 75-83% of the proteolytic activity of gut contents. Substrate SDS-polyacrylamide gel electrophoresis revealed nine different proteases ranging from 15 to 73 kDa.

  5. A practical total synthesis of the microbial alkaline proteinase inhibitor (MAPI).

    Science.gov (United States)

    Haebich, Dieter; Hillisch, Alexander; El Sheikh, Sherif

    2009-12-01

    Diverse serine and cysteine proteases as well as alkaline proteinases and elastases play a crucial role in numerous biological processes. Natural peptide aldehydes such as the "microbial alkaline proteinase inhibitor" (MAPI, 1) are valuable tools to characterize novel enzymes and to study their function in nature. Within a drug discovery program we wanted to design and explore non-natural MAPI congeners with novel biological profiles. To that end we devised a simple, practical, and scalable synthesis of MAPI 1 from readily available amino acid building blocks. The modular nature of our approach allows convenient structural modification of the MAPI backbone.

  6. Heterologous expression and purification of barley (Hordeum vulgare L.) cysteine protease in yeast

    DEFF Research Database (Denmark)

    Rosenkilde, Anne Lind; Dionisio, Giuseppe; Holm, Preben Bach

    2011-01-01

    The mobilization of protein during germination of barley seeds is essential and Cysteine Proteases accounts for more than 90 % of the total proteolytic activity in the degradation of barley seed storage proteins [1]. Cysteine proteases exist as pro-enzyme until activated through reduction...... of the active site cysteines and via removal of the pro-domain. The complement of cysteine proteases is comprehensive and for detailed studies of the individual components of this complement, a fast and efficient eukaryotic expression platform is highly desirable. The barley key cysteine protease, endoprotease...

  7. Searching for halo-alkalophilic proteases maintaining stability and activity in hydrophilic aprotic solvents as biocatalysts in carbohydrate chemistry

    DEFF Research Database (Denmark)

    Pedersen, Lars Haastrup; Mørkholt, Camilla Kær; Nielsen, Carsten Bue

    2012-01-01

    and other carbohydrates (1, 2). Therefore, we are searching for new bacterial halo-alkaline proteases from extreme environments in Denmark. So far we have identified a number of interesting isolates showing proteolytic activity at pH 7 and 10. Whole genome Illumina Hiseq 2000 amplicon sequencing, de novo...... assembly of the genome and automatic annotation of genes in the genome was carried out by RAST to find the genes responsible for protease production. The amino-acid sequence of the expressed proteolytic proteins was analyzed using tandem MS. The isolates identified so far belong to Bacillus sp. One...... of the interesting genomes with a total length of 4.1 Mb was assembled in 42 contigs with a total of 3070 known genes of which 39 are encoding for proteases (one serine endopeptidase). Another genome was assembled in 219 contigs with 4004 coding sequences and 2821 known genes of which 36 are encoding for proteases...

  8. Comparative analysis on the distribution of protease activities among fruits and vegetable resources.

    Science.gov (United States)

    Sun, Qian; Zhang, Bin; Yan, Qiao-Juan; Jiang, Zheng-Qiang

    2016-12-15

    In this study, a comparative analysis on the distribution of protease activities among 90 plant resources, including fruits and vegetables, has been performed. Protease activities of plant extracts were assayed at different pH values (pH 3.0, pH 7.5 and pH 10.5) using casein as a substrate. Ten fruits and thirteen vegetables show protease activities above 10U/g. Pineapple, fig and papaya, which are used for commercial protease production, exhibited high protease activities. Additionally, high protease activities were detected in kiwifruit (28.8U/g), broccoli (16.9U/g), ginger (16.6U/g), leek (32.7U/g) and red pepper (15.8U/g) at different pH values. SDS-PAGE and zymograms confirmed that various types of proteases existed in the five plant extracts and might be explored. Furthermore, five plant extracts were treated by different protease inhibitors. These results show that there are still many plant resources unexplored, which may be promising candidates for plant-derived protease production.

  9. Proteases from Latex of Euphorbia spp. and Its Application on Milk Clot Formation

    Directory of Open Access Journals (Sweden)

    Fidia Fibriana

    2015-09-01

    Full Text Available Crude proteases were extracted from Euphorbiaceae family, i.e. E. milii var imperata, E. trigona, and E. maculata. Among those three crude proteases, the activity of protease from E. trigona was the highest (812.50 U/ml, whereas E. milii and E. maculata crude proteases activity were 298.60 U/ml and 95.80 U/ml, respectively. E. maculata protein concentration was the highest among those three crude enzymes (1.206 mg/ml. The optimum pH and temperature of the enzymes were pH 7.0, pH 6.0, pH 6.5 and 60 °C, 50 °C, and 50 °C, respectively. Crude protease from E. milii var imperata, E. trigona, and E. maculata retained proteolytic activity over a wide range of pH (5.0–9.0 and temperature (up to 65 °C with casein as substrate. All crude proteases showed milk clotting activity ranged from 0.58 U/ml to 1.01 U/ml. Thus, these crude proteases are potential to be applied in dairy industries. However, further study on enzyme purification and characterization are necessary to obtain high purity of proteases before its application.Protease kasar berhasil diekstrak dari tanaman family Euphorbiaceae, yaitu E. milii var imperata, E. trigona, dan E. maculata. Diantara ketiga protease tersebut, aktivitas protease tertinggi diperoleh dari E. trigona (812,50 U/ml, sedangkan aktivitas protease dari E. milii dan E. maculata adalah 298,60 U/ml dan 95,80 U/ml, berturut-turut. Konsentrasi total protein tertinggi terdapat pada protease kasar E. maculata (1,206 mg/ml. pH dan suhu optimum ketiga enzim tersebut adalah pH 7.0, pH 6.0, pH 6.5 dan suhu 60 °C, 50 °C, and 50 °C, berturut-turut. Protease kasar dari E. milii var imperata, E. trigona, dan E. maculata menunjukkan aktivitas proteolitik pada rentang pH 5.0–9.0 dan rentang suhu sampai 65 °C menggunakan kasein sebagai substrat. Semua protease kasar menunjukkan aktivitas penggumpalan susu dengan rentang dari 0,58 U/ml sampai 1,01 U/ml. Berdasarkan hasil yang diperoleh, protease kasar dari ketiga jenis tanaman ini

  10. Optimization of Soilless Media for Alkaline Irrigation Water

    OpenAIRE

    Tramp, Cody Alexander; Chard, Julie K.; Bugbee, Bruce

    2009-01-01

    High root zone pH reduces nutrient availability and high alkalinity water is strongly buffered around an alkaline pH. Soilless media can be altered to improve nutrient availability. This study was conducted to optimize the composition of soilless media for use with high alkalinity water. Mixes of peat and/or perlite or vermiculite in 50/50 and 33/33/33 volumetric ratios were tested. In some studies, mixes were also amended with up to 2.4 g/L of dolomite limestone to neutralize the initial aci...

  11. 凡纳滨对虾虾头内源性蛋白酶同源性分析%Homology Analysis of Endogenous Protease from the Head of Litopenaeus Vannamei Shrimp

    Institute of Scientific and Technical Information of China (English)

    庄志凯; 杜嵇华

    2012-01-01

    Endogenous alkaline protease from the head of Litopenaeus vannamei shrimp was purified by Q-Sepharose FF and SephadexG-150 chromatography. SDS-PAGE showed a single band of 79. 95 kD. Endogenous acidic protease was purified by DEAE-Sepharose FF and SephadexG-100 chromatography. And SDS-PAGE showed a single band at molecular weight of 27. 45 kD. The ammo acid sequences were detected by reversed-phase high-performance liquid chromatography with electrospray ionization tandem mass spectrometry ( HPLC-ESI-MS/MS). The trypsin and pepsin digestion profiles were identified through matching NCBI database. The sequence alignment of the two proteases with proteases obtained from other species was performed using Vector NTI suite 8. 0. A LSSPATLVSRVATVSLPR.segment in the alkaline protease was found to be homologous to trypsin from pig. A terminal EFGLSETEPGTNF segment of the acidic protease was homologous to gastricsin from Xenopus laevis.%以凡纳滨对虾(Litopenaeus vannamei shrimp)虾头为原料,采用Q- Sepharose F F和Sephadex G-150对虾头内源碱性蛋白酶进行了纯化,通过SDS-PAGE测定分子量为79.95 kD;采用DEAE-Sepharose F.F和Sephadex G-100对内源酸性蛋白酶进行了纯化,通过SDS-PAGE测定分子量为27.45 kD.利用HPLC-ESI-MS/MS对虾头内源碱性和酸性蛋白酶同源性进行了初步分析,将检测到内源性蛋白酶的部分氨基酸序列分别与不同物种的胰蛋白酶和胃蛋白酶氨基酸序列于Vector NTI suite 8.0软件上进行序列比对.结果表明,内源性碱性蛋白酶与猪胰蛋白酶具有很高的同源性,均含有氨基酸序列LSSPATLNSRVATVSLPR;内源性酸性蛋白酶与非洲蟾蜍胃亚蛋白酶具有很高的同源性,均含有氨基酸序列EFGLSETEPGTNF.

  12. CHARACTERIZATION OF PROTEASE PRODUCED BY “JINHUA”FUNGI FROM FUZHUAN BRICK TEA%茯砖茶中“金花”菌产蛋白酶特性的研究

    Institute of Scientific and Technical Information of China (English)

    丁婷; 吕嘉枥; 侯蓓

    2011-01-01

    在不同培养时间段对“金花”菌产酸、中、碱性蛋白酶酶活进行测定,研究了不同碳源、氮源、无机盐等培养条件对产中性及碱性蛋白酶酶活的影响.结果表明:“金花”菌所产蛋白酶以中性和碱性蛋白酶为主,最适碳源为乳糖,氮源为麸皮,最适无机盐为氯化钠.在此条件下 “金花”菌产中性蛋白酶的酶活可达到237 U/g,产碱性蛋白酶的酶活可达236 U/g.“金花”菌对茯砖茶的消化作用有促进作用.%The acid protease activity, neutral protease and alkaline protease which produced by "Jinhua" fungi was measured at different cultural time. The effect of addition of carbon sources, nitrogen sources and inorganic salts on producing neutral and alkaline protease was studied. The results show that the many produced protease of "Jinhua" fungi was neutral protease and alkaline protease. Carbon source and nitrogen source preferred for protease production were lactose and bran, while inorganic salts preferred were sodium chloride. Under these conditions, the neutral protease activity was 237 U/g and the alkaline protease activity was 236 U/g.

  13. Curcumin derivatives as HIV-1 protease inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Sui, Z.; Li, J.; Craik, C.S.; Ortiz de Montellano, P.R. [Univ. of California, San Francisco, CA (United States)

    1993-12-31

    Curcumin, a non-toxic natural compound from Curcuma longa, has been found to be an HIV-1 protease inhibitor. Some of its derivatives were synthesized and their inhibitory activity against the HIV-1 protease was tested. Curcumin analogues containing boron enhanced the inhibitory activity. At least of the the synthesized compounds irreversibly inhibits the HIV-1 protease.

  14. Space-time variability of alkalinity in the Mediterranean Sea

    Directory of Open Access Journals (Sweden)

    G. Cossarini

    2014-09-01

    Full Text Available The paper provides a basin assessment of the spatial distribution of ocean alkalinity in the Mediterranean Sea. The assessment is made using a 3-D transport-biogeochemical-carbonate model to integrate the available experimental findings, which also constrains model output. The results indicate that the Mediterranean Sea shows alkalinity values that are much higher than those observed in the Atlantic Ocean on a basin-wide scale. A marked west-to-east surface gradient of alkalinity is reproduced as a response to the terrestrial discharges, the mixing effect with the Atlantic water entering from the Gibraltar Strait and the Black Sea water from Dardanelles, and the surface flux of evaporation minus precipitation. Dense water production in marginal seas (Adriatic and Aegean Seas, where alkaline inputs are relevant, and the Mediterranean thermohaline circulation sustains the west-to-east gradient along the entire water column. In the surface layers, alkalinity has a relevant seasonal cycle (up to 40 μmol kg−1 that is driven both by physical and biological processes. A comparison of alkalinity vs. salinity indicates that different regions present different relationships. In regions of freshwater influence, the two measures are negatively correlated due to riverine alkalinity input, whereas they are positively correlated in open seas. Alkalinity always is much higher than in the Atlantic waters, which might indicate a higher than usual buffering capacity towards ocean acidification, even at high concentrations of dissolved inorganic carbon.

  15. Response of Desulfovibrio vulgaris to Alkaline Stress

    Energy Technology Data Exchange (ETDEWEB)

    Stolyar, S.; He, Q.; He, Z.; Yang, Z.; Borglin, S.E.; Joyner, D.; Huang, K.; Alm, E.; Hazen, T.C.; Zhou, J.; Wall, J.D.; Arkin, A.P.; Stahl, D.A.

    2007-11-30

    The response of exponentially growing Desulfovibrio vulgarisHildenborough to pH 10 stress was studied using oligonucleotidemicroarrays and a study set of mutants with genes suggested by microarraydata to be involved in the alkaline stress response deleted. The datashowed that the response of D. vulgaris to increased pH is generallysimilar to that of Escherichia coli but is apparently controlled byunique regulatory circuits since the alternative sigma factors (sigma Sand sigma E) contributing to this stress response in E. coli appear to beabsent in D. vulgaris. Genes previously reported to be up-regulated in E.coli were up-regulated in D. vulgaris; these genes included three ATPasegenes and a tryptophan synthase gene. Transcription of chaperone andprotease genes (encoding ATP-dependent Clp and La proteases and DnaK) wasalso elevated in D. vulgaris. As in E. coli, genes involved in flagellumsynthesis were down-regulated. The transcriptional data also identifiedregulators, distinct from sigma S and sigma E, that are likely part of aD. vulgaris Hildenborough-specific stress response system.Characterization of a study set of mutants with genes implicated inalkaline stress response deleted confirmed that there was protectiveinvolvement of the sodium/proton antiporter NhaC-2, tryptophanase A, andtwo putative regulators/histidine kinases (DVU0331 andDVU2580).

  16. Production of an extensive sunflower protein hydrolysate by sequential hydrolysis with endo- and exo-proteases.

    Directory of Open Access Journals (Sweden)

    Villanueva, Alvaro

    1999-12-01

    Full Text Available A high quality protein isolate has been obtained from defatted sunflower meal by alkaline extraction and isoelectric precipitation. Protein content was increased from 31.2 % in the defatted flour to 97 % in the protein isolate. The percentages of fiber, soluble sugars, polyphenols and residual lipids in the protein isolate were reduced to more than 90 % with respect to the defatted meal. The protein isolate was used as starting material for the generation of an extensive enzymatic protein hydrolysate. The hydrolysis was carried out in a pH stat using sequentially an endo-protease (Alcalase and an exo-protease (Flavourzyme. The protein hydrolysate, with a degree of hydrolysis of 50.7 %, was white and non bitter.

    Se ha obtenido un aislado proteico de alta calidad a partir de harina desengrasada de girasol, mediante extracción alcalina y precipitación isoeléctrica. Se incrementó el contenido proteico desde un 31.2 % en la harina desengrasada hasta un 97 % en el aislado proteico. Los porcentajes de fibra, azúcares solubles, polifenoles y lípidos residuales se redujeron en más del 90 % en el aislado proteico respecto a la harina desengrasada. Se usó el aislado proteico como material de partida para la producción de un hidrolizado enzimático proteico extenso. La hidrólisis se realizó en un reactor usando secuencialmente una endo-proteasa (Alcalasa y una exo-proteasa (Flavorzima. El hidrolizado proteico, con un grado de hidrólisis del 50.7 %, era blanco y no presentaba amargor.

  17. Use Alkalinity Monitoring to Optimize Bioreactor Performance.

    Science.gov (United States)

    Jones, Christopher S; Kult, Keegan J

    2016-05-01

    In recent years, the agricultural community has reduced flow of nitrogen from farmed landscapes to stream networks through the use of woodchip denitrification bioreactors. Although deployment of this practice is becoming more common to treat high-nitrate water from agricultural drainage pipes, information about bioreactor management strategies is sparse. This study focuses on the use of water monitoring, and especially the use of alkalinity monitoring, in five Iowa woodchip bioreactors to provide insights into and to help manage bioreactor chemistry in ways that will produce desirable outcomes. Results reported here for the five bioreactors show average annual nitrate load reductions between 50 and 80%, which is acceptable according to established practice standards. Alkalinity data, however, imply that nitrous oxide formation may have regularly occurred in at least three of the bioreactors that are considered to be closed systems. Nitrous oxide measurements of influent and effluent water provide evidence that alkalinity may be an important indicator of bioreactor performance. Bioreactor chemistry can be managed by manipulation of water throughput in ways that produce adequate nitrate removal while preventing undesirable side effects. We conclude that (i) water should be retained for longer periods of time in bioreactors where nitrous oxide formation is indicated, (ii) measuring only nitrate and sulfate concentrations is insufficient for proper bioreactor operation, and (iii) alkalinity monitoring should be implemented into protocols for bioreactor management.

  18. Application of Alkaline Waterflooding to a High Acidity Crude Oil Application de l'injection d'eau alcaline au cas d'un pétrole brut à forte acidité

    Directory of Open Access Journals (Sweden)

    Abdel-Waly A.

    2006-11-01

    Full Text Available The main objective of this work was to study the enhanced recovery of a high acidity crude oil (South Geisum crude by alkaline solutions. Different properties of South Geisum crude oil, namely acidity, interfacial tension, and contact angle, were investigated. Displacement tests were carried out to study the effect of alkaline slug concentration, slug size, oil alkali type, and temperature viscosity on recovery. South Geisum crude oil is a highly acidic crude (4. 38 mg KOH/g. It was found that the interfacial tension between crude oil and formation water decreases with increasing alkaline concentration until it reaches a minimum, after which it increases again with a further increase in alkaline concentration. Interfacial tension between crude oil and displacement water also decreases with increasing alkaline concentration. Contact angle measurements indicated oil-wetting conditions that increase by the addition of alkaline solutions. Displacement floods showed that, at the early stages of displacement, oil recovery increases with increasing alkaline concentration until it reaches a maximum at 4 % by weight NaOH concentration. Also, at such early stages, an excessive increase in alkaline concentration results in lower oil recovery. On the other hand, after the injection of many pore volumes of water, oil recovery is almost the same regardless of the alkaline concentration. It was found also that oil recovery increases with increasing alkaline slug size until it reaches a maximum at 15 % PV, after which increasing slug size results in decreasing oil recovery (this result has not as yet been reported in the literature. Sodium hydroxide slugs produce more oil recovery than sodium carbonate slugs. Oil recovery increases with increasing temperature (from 25 to 55°C and decreasing oil viscosity. Cet article traite de la récupération, au moyen de solutions alcalines, d'un pétrole brut à forte acidité (brut de Geisum-Sud. Différentes propri

  19. Novel As-doped, As and N-codoped carbon nanotubes as highly active and durable electrocatalysts for O2 reduction in alkaline medium

    Science.gov (United States)

    Liu, Ziwu; Li, Meng; Wang, Fang; Wang, Quan-De

    2016-02-01

    To develop more efficient metal-free cathode electrocatalysts for fuel cells, novel arsenic (As)-doped, As and N-codoped carbon nanotubes are synthesized by chemical vapor deposition in this work. The as-prepared As-containing carbon nanotubes exhibit significantly enhanced activity and long-term durability for the oxygen reduction reaction (ORR) in alkaline medium, indicating that the doping of As or codoping As with other heteroatoms into carbon matrix could improve the ORR activity of carbon materials due to the changes in electronic and physical properties of carbon nanotubes evidenced by density functional theory calculations. Moreover, As-containing carbon nanotubes also display much better methanol tolerance, showing a good potential application for future fuel cells.

  20. Protease-specific nanosensors for magnetic resonance imaging.

    Science.gov (United States)

    Schellenberger, Eyk; Rudloff, Franziska; Warmuth, Carsten; Taupitz, Matthias; Hamm, Bernd; Schnorr, Jörg

    2008-12-01

    Imaging of enzyme activity is a central goal of molecular imaging. With the introduction of fluorescent smart probes, optical imaging has become the modality of choice for experimental in vivo detection of enzyme activity. Here, we present a novel high-relaxivity nanosensor that is suitable for in vivo imaging of protease activity by magnetic resonance imaging. Upon specific protease cleavage, the nanoparticles rapidly switch from a stable low-relaxivity stealth state to become adhesive, aggregating high-relaxivity particles. To demonstrate the principle, we chose a cleavage motif of matrix metalloproteinase 9 (MMP-9), an enzyme important in inflammation, atherosclerosis, tumor progression, and many other diseases with alterations of the extracellular matrix. On the basis of clinically tested very small iron oxide particles (VSOP), the MMP-9-activatable protease-specific iron oxide particles (PSOP) have a hydrodynamic diameter of only 25 nm. PSOP are rapidly activated, resulting in aggregation and increased T2*-relaxivity.

  1. Peptide synthesis in neat organic solvents with novel thermostable proteases.

    Science.gov (United States)

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

    2015-06-01

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

  2. The biology of cystatin M/E and its cognate target proteases.

    NARCIS (Netherlands)

    Zeeuwen, P.L.J.M.; Cheng, T.; Schalkwijk, J.

    2009-01-01

    Cystatin M/E is a member of a superfamily of evolutionarily-related cysteine protease inhibitors that provide regulatory and protective functions against uncontrolled proteolysis by cysteine proteases. Although most cystatins are ubiquitously expressed, high levels of cystatin M/E expression are mai

  3. Mitochondrial cereblon functions as a Lon-type protease.

    Science.gov (United States)

    Kataoka, Kosuke; Nakamura, China; Asahi, Toru; Sawamura, Naoya

    2016-07-15

    Lon protease plays a major role in the protein quality control system in mammalian cell mitochondria. It is present in the mitochondrial matrix, and degrades oxidized and misfolded proteins, thereby protecting the cell from various extracellular stresses, including oxidative stress. The intellectual disability-associated and thalidomide-binding protein cereblon (CRBN) contains a large, highly conserved Lon domain. However, whether CRBN has Lon protease-like function remains unknown. Here, we determined if CRBN has a protective function against oxidative stress, similar to Lon protease. We report that CRBN partially distributes in mitochondria, suggesting it has a mitochondrial function. To specify the mitochondrial role of CRBN, we mitochondrially expressed CRBN in human neuroblastoma SH-SY5Y cells. The resulting stable SH-SY5Y cell line showed no apparent effect on the mitochondrial functions of fusion, fission, and membrane potential. However, mitochondrially expressed CRBN exhibited protease activity, and was induced by oxidative stress. In addition, stably expressed cells exhibited suppressed neuronal cell death induced by hydrogen peroxide. These results suggest that CRBN functions specifically as a Lon-type protease in mitochondria.

  4. Biotechnology of cold-active proteases.

    Science.gov (United States)

    Joshi, Swati; Satyanarayana, Tulasi

    2013-05-03

    The bulk of Earth's biosphere is cold (<5 °C) and inhabited by psychrophiles. Biocatalysts from psychrophilic organisms (psychrozymes) have attracted attention because of their application in the ongoing efforts to decrease energy consumption. Proteinases as a class represent the largest category of industrial enzymes. There has been an emphasis on employing cold-active proteases in detergents because this allows laundry operations at ambient temperatures. Proteases have been used in environmental bioremediation, food industry and molecular biology. In view of the present limited understanding and availability of cold-active proteases with diverse characteristics, it is essential to explore Earth's surface more in search of an ideal cold-active protease. The understanding of molecular and mechanistic details of these proteases will open up new avenues to tailor proteases with the desired properties. A detailed account of the developments in the production and applications of cold-active proteases is presented in this review.

  5. Biotechnology of Cold-Active Proteases

    Directory of Open Access Journals (Sweden)

    Tulasi Satyanarayana

    2013-05-01

    Full Text Available The bulk of Earth’s biosphere is cold (<5 °C and inhabited by psychrophiles. Biocatalysts from psychrophilic organisms (psychrozymes have attracted attention because of their application in the ongoing efforts to decrease energy consumption. Proteinases as a class represent the largest category of industrial enzymes. There has been an emphasis on employing cold-active proteases in detergents because this allows laundry operations at ambient temperatures. Proteases have been used in environmental bioremediation, food industry and molecular biology. In view of the present limited understanding and availability of cold-active proteases with diverse characteristics, it is essential to explore Earth’s surface more in search of an ideal cold-active protease. The understanding of molecular and mechanistic details of these proteases will open up new avenues to tailor proteases with the desired properties. A detailed account of the developments in the production and applications of cold-active proteases is presented in this review.

  6. Pyrosequencing reveals the key microorganisms involved in sludge alkaline fermentation for efficient short-chain fatty acids production.

    Science.gov (United States)

    Zheng, Xiong; Su, Yinglong; Li, Xiang; Xiao, Naidong; Wang, Dongbo; Chen, Yinguang

    2013-05-07

    Short-chain fatty acids (SCFAs) have been regarded as the excellent carbon source of wastewater biological nutrient removal, and sludge alkaline (pH 10) fermentation has been reported to achieve highly efficient SCFAs production. In this study, the underlying mechanisms for the improved SCFAs production at pH 10 were investigated by using 454 pyrosequencing and fluorescent in situ hybridization (FISH) to analyze the microbial community structures in sludge fermentation reactors. It was found that sludge fermentation at pH 10 increased the abundances of Pseudomonas sp. and Alcaligenes sp., which were able to excrete extracellular proteases and depolymerases, and thus enhanced the hydrolysis of insoluble sludge protein and polyhydroxyalkanoates (PHA). Meanwhile, the abundance of acid-producing bacteria (such as Clostridium sp.) in the reactor of pH 10 was also higher than that of uncontrolled pH, which benefited the acidification of soluble organic substrates. Further study indicated that sludge fermentation at pH 10 significantly decreased the number of methanogenic archaea, resulting in lower SCFAs consumption and lower methane production. Therefore, anaerobic sludge fermentation under alkaline conditions increased the abundances of bacteria involved in sludge hydrolysis and acidification, and decreased the abundance of methanogenic archaea, which favored the competition of bacteria over methanogens and resulted in the efficient production of SCFAs.

  7. Extracellular proteases of Halobacillus blutaparonensis strain M9, a new moderately halophilic bacterium

    Science.gov (United States)

    Santos, Anderson F.; Valle, Roberta S.; Pacheco, Clarissa A.; Alvarez, Vanessa M.; Seldin, Lucy; Santos, André L.S.

    2013-01-01

    Halophilic microorganisms are source of potential hydrolytic enzymes to be used in industrial and/or biotechnological processes. In the present study, we have investigated the ability of the moderately halophilic bacterium Halobacillus blutaparonensis (strain M9), a novel species described by our group, to release proteolytic enzymes. This bacterial strain abundantly proliferated in Luria-Bertani broth supplemented with 2.5% NaCl as well as secreted proteases to the extracellular environment. The production of proteases occurred in bacterial cells grown under different concentration of salt, ranging from 0.5% to 10% NaCl, in a similar way. The proteases secreted by H. blutaparonensis presented the following properties: (i) molecular masses ranging from 30 to 80 kDa, (ii) better hydrolytic activities under neutral-alkaline pH range, (iii) expression modulated according to the culture age, (iv) susceptibility to phenylmethylsulphonyl fluoride, classifying them as serine-type proteases, (v) specific cleavage over the chymotrypsin substrate, and (vi) enzymatic stability in the presence of salt (up to 20% NaCl) and organic solvents (e.g., ether, isooctane and cyclohexane). The proteases described herein are promising for industrial practices due to its haloalkaline properties. PMID:24688526

  8. Extracellular proteases of Halobacillus blutaparonensis strain M9, a new moderately halophilic bacterium

    Directory of Open Access Journals (Sweden)

    Anderson F. Santos

    2013-12-01

    Full Text Available Halophilic microorganisms are source of potential hydrolytic enzymes to be used in industrial and/or biotechnological processes. In the present study, we have investigated the ability of the moderately halophilic bacterium Halobacillus blutaparonensis (strain M9, a novel species described by our group, to release proteolytic enzymes. This bacterial strain abundantly proliferated in Luria-Bertani broth supplemented with 2.5% NaCl as well as secreted proteases to the extracellular environment. The production of proteases occurred in bacterial cells grown under different concentration of salt, ranging from 0.5% to 10% NaCl, in a similar way. The proteases secreted by H. blutaparonensis presented the following properties: (i molecular masses ranging from 30 to 80 kDa, (ii better hydrolytic activities under neutral-alkaline pH range, (iii expression modulated according to the culture age, (iv susceptibility to phenylmethylsulphonyl fluoride, classifying them as serine-type proteases, (v specific cleavage over the chymotrypsin substrate, and (vi enzymatic stability in the presence of salt (up to 20% NaCl and organic solvents (e.g., ether, isooctane and cyclohexane. The proteases described herein are promising for industrial practices due to its haloalkaline properties.

  9. Modeling and structural analysis of PA clan serine proteases

    Directory of Open Access Journals (Sweden)

    Laskar Aparna

    2012-05-01

    Full Text Available Abstract Background Serine proteases account for over a third of all known proteolytic enzymes; they are involved in a variety of physiological processes and are classified into clans sharing structural homology. The PA clan of endopeptidases is the most abundant and over two thirds of this clan is comprised of the S1 family of serine proteases, which bear the archetypal trypsin fold and have a catalytic triad in the order Histidine, Aspartate, Serine. These proteases have been studied in depth and many three dimensional structures have been experimentally determined. However, these structures mostly consist of bacterial and animal proteases, with a small number of plant and fungal proteases and as yet no structures have been determined for protozoa or archaea. The core structure and active site geometry of these proteases is of interest for many applications. This study investigated the structural properties of different S1 family serine proteases from a diverse range of taxa using molecular modeling techniques. Results Our predicted models from protozoa, archaea, fungi and plants were combined with the experimentally determined structures of 16 S1 family members and used for analysis of the catalytic core. Amino acid sequences were submitted to SWISS-MODEL for homology-based structure prediction or the LOOPP server for threading-based structure prediction. Predicted models were refined using INSIGHT II and SCRWL and validated against experimental structures. Investigation of secondary structures and electrostatic surface potential was performed using MOLMOL. The structural geometry of the catalytic core shows clear deviations between taxa, but the relative positions of the catalytic triad residues were conserved. Some highly conserved residues potentially contributing to the stability of the structural core were identified. Evolutionary divergence was also exhibited by large variation in secondary structure features outside the core

  10. The binding mechanism of a peptidic cyclic serine protease inhibitor

    DEFF Research Database (Denmark)

    Jiang, Longguang; Svane, Anna S P; Sørensen, Hans Peter

    2011-01-01

    Serine proteases are classical objects for studies of catalytic and inhibitory mechanisms as well as interesting as therapeutic targets. Since small-molecule serine protease inhibitors generally suffer from specificity problems, peptidic inhibitors, isolated from phage-displayed peptide libraries...... inhibitory mechanism and an unusually high specificity. Using a number of modified variants of upain-1, we characterised the upain-1-urokinase-type plasminogen activator complex using X-ray crystal structure analysis, determined a model of the peptide in solution by NMR spectroscopy, and analysed binding...... kinetics and thermodynamics by surface plasmon resonance and isothermal titration calorimetry. We found that upain-1 changes both main-chain conformation and side-chain orientations as it binds to the protease, in particular its Trp3 residue and the surrounding backbone. The properties of upain-1...

  11. New therapeutic strategies in HCV: second-generation protease inhibitors.

    Science.gov (United States)

    Clark, Virginia C; Peter, Joy A; Nelson, David R

    2013-02-01

    Telaprevir and boceprevir are the first direct-acting antiviral agents approved for use in HCV treatment and represent a significant advance in HCV therapy. However, these first-generation drugs also have significant limitations related to thrice-daily dosing, clinically challenging side-effect profiles, low barriers to resistance and a lack of pan-genotype activity. A second wave of protease inhibitors are in phase II and III trials and promise to provide a drug regimen with a better dosing schedule and improved tolerance. These second-wave protease inhibitors will probably be approved in combination with PEG-IFN and Ribavirin (RBV), as well as future all-oral regimens. The true second-generation protease inhibitors are in earlier stages of development and efficacy data are anxiously awaited as they may provide pan-genotypic antiviral activity and a high genetic barrier to resistance. © 2012 John Wiley & Sons A/S.

  12. Alkaline chemistry of transuranium elements and technetium and the treatment of alkaline radioactive wastes

    Energy Technology Data Exchange (ETDEWEB)

    Delegard, C.H. [Westinghouse Hanford Co., Richland, WA (United States); Peretrukhin, V.F.; Shilov, V.P.; Pikaev, A.K. [Russian Academy of Sciences (Russian Federation). Inst. of Physical Chemistry

    1995-05-01

    Goal of this survey is to generalize the known data on fundamental physical-chemical properties of TRUs and Tc, methods for their isolation, and to provide recommendations that will be useful for partitioning them from alkaline high-level wastes.

  13. Isolation and partial characterization of a protease from Agave americana variegata.

    Science.gov (United States)

    Du Toit, P J

    1976-05-13

    A new protease was isolated from an extract of leaves of Agave americana variegata. The protease (EC 3.4.-) was purified 565-fold with a yield of 39.5%. The 43.8 mg enzyme had a specific activity of 0.44 units/mg. According to electrophoretic, ultracentrifugal and other physical characterizations the enzyme was homogeneous. The enzyme had a MR of 57000, a S20,W-value of 4.37 S, a D20, W-value of 6.8-7.0 - 10(-7) cm2sec-1, a Stokes radius of 3.18 nm, a partial specific volume of 0.735 cm3g-1, a frictional ration of 1.25, a molecular absorbancy index at 280 nm of 5.773-10(4), an isoelectric point of 5.25 and contained 8-10% carbohydrate. The enzyme contained no cysteine. Agave protease could hydrolyze a variety of protein substrates although it did have a restricted specificity. It is not a sulphhydryl protease but seems to be an alkaline "serine" protease with an optimum pH of 7.8-8.0 Agave protease had marked esterolytic activity and with Cbz-Tyr-ONp had an apparent Michaelis constant of 0.0345 -10(-3) M and a V of 1.24 mol substrate/mol enzyme per sec. The enzyme did not need metal ions for optimal activity, monovalent cations did not influence its kinetic parameters, but it was inhibited by cobalt, pC1HgBzO- and TosPheCH2C1. With respect to its primary specificity, as well as its pH-dependence there was a resemblance with chymotrypsin, although the rate of hydrolysis of Agave protease is much lower.

  14. Further characterization of a highly attenuated Yersinia pestis CO92 mutant deleted for the genes encoding Braun lipoprotein and plasminogen activator protease in murine alveolar and primary human macrophages.

    Science.gov (United States)

    van Lier, Christina J; Tiner, Bethany L; Chauhan, Sadhana; Motin, Vladimir L; Fitts, Eric C; Huante, Matthew B; Endsley, Janice J; Ponnusamy, Duraisamy; Sha, Jian; Chopra, Ashok K

    2015-03-01

    We recently characterized the Δlpp Δpla double in-frame deletion mutant of Yersinia pestis CO92 molecularly, biologically, and immunologically. While Braun lipoprotein (Lpp) activates toll-like receptor-2 to initiate an inflammatory cascade, plasminogen activator (Pla) protease facilitates bacterial dissemination in the host. The Δlpp Δpla double mutant was highly attenuated in evoking bubonic and pneumonic plague, was rapidly cleared from mouse organs, and generated humoral and cell-mediated immune responses to provide subsequent protection to mice against a lethal challenge dose of wild-type (WT) CO92. Here, we further characterized the Δlpp Δpla double mutant in two murine macrophage cell lines as well as in primary human monocyte-derived macrophages to gauge its potential as a live-attenuated vaccine candidate. We first demonstrated that the Δpla single and the Δlpp Δpla double mutant were unable to survive efficiently in murine and human macrophages, unlike WT CO92. We observed that the levels of Pla and its associated protease activity were not affected in the Δlpp single mutant, and, likewise, deletion of the pla gene from WT CO92 did not alter Lpp levels. Further, our study revealed that both Lpp and Pla contributed to the intracellular survival of WT CO92 via different mechanisms. Importantly, the ability of the Δlpp Δpla double mutant to be phagocytized by macrophages, to stimulate production of tumor necrosis factor-α and interleukin-6, and to activate the nitric oxide killing pathways of the host cells remained unaltered when compared to the WT CO92-infected macrophages. Finally, macrophages infected with either the WT CO92 or the Δlpp Δpla double mutant were equally efficient in their uptake of zymosan particles as determined by flow cytometric analysis. Overall, our data indicated that although the Δlpp Δpla double mutant of Y. pestis CO92 was highly attenuated, it retained the ability to elicit innate and subsequent acquired immune

  15. Simultaneous determination of 24 or more acidic and alkaline phytohormones in femtomole quantities of plant tissues by high-performance liquid chromatography-electrospray ionization-ion trap mass spectrometry.

    Science.gov (United States)

    Liu, Shichang; Chen, Weiqi; Qu, Long; Gai, Ying; Jiang, Xiangning

    2013-02-01

    Phytohormones act at relatively low concentrations as major regulatory factors of plant growth and development, and cross talk of phytohormones is currently of great interest throughout the plant science community. To meet this demand, a method that is capable of simultaneously analyzing diverse plant hormones is essential. This paper introduces a high-performance liquid chromatographic separation technique coupled with sensitive and selective ion trap mass spectrometry to simultaneously determine 24 or more acidic and alkaline phytohormones, including auxin, cis- and trans-abscisic acid, 11 cytokinins, and 10 gibberellins, in a single injection of sample. A binary solid-phase extraction using Oasis MCX cartridges for cations and Oasis MAX cartridges for anions was used to prepurify more than 24 acidic and alkaline phytohormones from a single plant extract. The method showed good linearity for all 24 phytohormones with R(2) values ranging from 0.9903 to 0.9997. Limits of detection for most of the phytohormones were in the femtomole range with some extending into the sub-femtomole range. This method was applied to hundreds of plant samples comprising different tissues from various plants, including herbaceous, woody climbing, and woody plants to demonstrate feasibility and to validate the methodology.

  16. Protease-sensitive synthetic prions.

    Directory of Open Access Journals (Sweden)

    David W Colby

    2010-01-01

    Full Text Available Prions arise when the cellular prion protein (PrP(C undergoes a self-propagating conformational change; the resulting infectious conformer is designated PrP(Sc. Frequently, PrP(Sc is protease-resistant but protease-sensitive (s prions have been isolated in humans and other animals. We report here that protease-sensitive, synthetic prions were generated in vitro during polymerization of recombinant (rec PrP into amyloid fibers. In 22 independent experiments, recPrP amyloid preparations, but not recPrP monomers or oligomers, transmitted disease to transgenic mice (n = 164, denoted Tg9949 mice, that overexpress N-terminally truncated PrP. Tg9949 control mice (n = 174 did not spontaneously generate prions although they were prone to late-onset spontaneous neurological dysfunction. When synthetic prion isolates from infected Tg9949 mice were serially transmitted in the same line of mice, they exhibited sPrP(Sc and caused neurodegeneration. Interestingly, these protease-sensitive prions did not shorten the life span of Tg9949 mice despite causing extensive neurodegeneration. We inoculated three synthetic prion isolates into Tg4053 mice that overexpress full-length PrP; Tg4053 mice are not prone to developing spontaneous neurological dysfunction. The synthetic prion isolates caused disease in 600-750 days in Tg4053 mice, which exhibited sPrP(Sc. These novel synthetic prions demonstrate that conformational changes in wild-type PrP can produce mouse prions composed exclusively of sPrP(Sc.

  17. Inhibitors of lysosomal cysteine proteases

    Directory of Open Access Journals (Sweden)

    Lyanna O. L.

    2011-04-01

    Full Text Available The review is devoted to the inhibitors of cysteine proteinases which are believed to be very important in many biochemical processes of living organisms. They participate in the development and progression of numerous diseases that involve abnormal protein turnover. One of the main regulators of these proteinases is their specific inhibitors: cystatins. The aim of this review was to present current knowledge about endogenous inhibitors of lysosomal cysteine proteases and their synthetic analogs.

  18. Nelfinavir: fourth protease inhibitor approved.

    Science.gov (United States)

    1997-01-01

    The Food and Drug Administration (FDA) has granted accelerated approval to nelfinavir in both adult and pediatric formulations. Agouron, the manufacturer, used innovative computerized drug design techniques to discover, design, and refine the nelfinavir molecule. Nelfinavir is marketed under the trade name Viracept, and costs $5,000 per year. Early clinical trials find it to be as powerful as the other protease inhibitors, but with a different resistance profile. The drug has relatively few drug indications; however, several compounds have been contraindicated.

  19. Conserved hydrogen bonds and water molecules in MDR HIV-1 protease substrate complexes

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Zhigang [Wayne State Univ., Detroit, MI (United States); Case Western Reserve Univ., Cleveland, OH (United States); Harbor Hospital Baltimore, MD (United States); Wang, Yong [Wayne State Univ., Detroit, MI (United States); Yedidi, Ravikiran S. [Wayne State Univ., Detroit, MI (United States); National Institutes of Health, Bethesda, MD (United States); Dewdney, Tamaria G. [Wayne State Univ., Detroit, MI (United States); Reiter, Samuel J. [Wayne State Univ., Detroit, MI (United States); Brunzelle, Joseph S. [Northwestern Univ. Feinberg School of Medicine, Chicago, IL (United States); Kovari, Iulia A. [Wayne State Univ., Detroit, MI (United States); Kovari, Ladislau C. [Wayne State Univ., Detroit, MI (United States)

    2012-12-19

    Success of highly active antiretroviral therapy (HAART) in anti-HIV therapy is severely compromised by the rapidly developing drug resistance. HIV-1 protease inhibitors, part of HAART, are losing their potency and efficacy in inhibiting the target. Multi-drug resistant (MDR) 769 HIV-1 protease (resistant mutations at residues 10, 36, 46, 54, 62, 63, 71, 82, 84, 90) was selected for the present study to understand the binding to its natural substrates. The nine crystal structures of MDR769 HIV-1 protease substrate hepta-peptide complexes were analyzed in order to reveal the conserved structural elements for the purpose of drug design against MDR HIV-1 protease. Our structural studies demonstrated that highly conserved hydrogen bonds between the protease and substrate peptides, together with the conserved crystallographic water molecules, played a crucial role in the substrate recognition, substrate stabilization and protease stabilization. Additionally, the absence of the key flap-ligand bridging water molecule might imply a different catalytic mechanism of MDR769 HIV-1 protease compared to that of wild type (WT) HIV-1 protease.

  20. Multisystemic functions of alkaline phosphatases.

    Science.gov (United States)

    Buchet, René; Millán, José Luis; Magne, David

    2013-01-01

    Human and mouse alkaline phosphatases (AP) are encoded by a multigene family expressed ubiquitously in multiple tissues. Gene knockout (KO) findings have helped define some of the precise exocytic functions of individual isozymes in bone, teeth, the central nervous system, and in the gut. For instance, deficiency in tissue-nonspecific alkaline phosphatase (TNAP) in mice (Alpl (-/-) mice) and humans leads to hypophosphatasia (HPP), an inborn error of metabolism characterized by epileptic seizures in the most severe cases, caused by abnormal metabolism of pyridoxal-5'-phosphate (the predominant form of vitamin B6) and by hypomineralization of the skeleton and teeth featuring rickets and early loss of teeth in children or osteomalacia and dental problems in adults caused by accumulation of inorganic pyrophosphate (PPi). Enzyme replacement therapy with mineral-targeting TNAP prevented all the manifestations of HPP in mice, and clinical trials with this protein therapeutic are showing promising results in rescuing life-threatening HPP in infants. Conversely, TNAP induction in the vasculature during generalized arterial calcification of infancy (GACI), type II diabetes, obesity, and aging can cause medial vascular calcification. TNAP inhibitors, discussed extensively in this book, are in development to prevent pathological arterial calcification. The brush border enzyme intestinal alkaline phosphatase (IAP) plays an important role in fatty acid (FA) absorption, in protecting gut barrier function, and in determining the composition of the gut microbiota via its ability to dephosphorylate lipopolysaccharide (LPS). Knockout mice (Akp3 (-/-)) deficient in duodenal-specific IAP (dIAP) become obese, and develop hyperlipidemia and hepatic steatosis when fed a high-fat diet (HFD). These changes are accompanied by upregulation in the jejunal-ileal expression of the Akp6 IAP isozyme (global IAP, or gIAP) and concomitant upregulation of FAT/CD36, a phosphorylated fatty acid

  1. Protease gene families in Populus and Arabidopsis

    Directory of Open Access Journals (Sweden)

    Jansson Stefan

    2006-12-01

    Full Text Available Abstract Background Proteases play key roles in plants, maintaining strict protein quality control and degrading specific sets of proteins in response to diverse environmental and developmental stimuli. Similarities and differences between the proteases expressed in different species may give valuable insights into their physiological roles and evolution. Results We have performed a comparative analysis of protease genes in the two sequenced dicot genomes, Arabidopsis thaliana and Populus trichocarpa by using genes coding for proteases in the MEROPS database 1 for Arabidopsis to identify homologous sequences in Populus. A multigene-based phylogenetic analysis was performed. Most protease families were found to be larger in Populus than in Arabidopsis, reflecting recent genome duplication. Detailed studies on e.g. the DegP, Clp, FtsH, Lon, rhomboid and papain-Like protease families showed the pattern of gene family expansion and gene loss was complex. We finally show that different Populus tissues express unique suites of protease genes and that the mRNA levels of different classes of proteases change along a developmental gradient. Conclusion Recent gene family expansion and contractions have made the Arabidopsis and Populus complements of proteases different and this, together with expression patterns, gives indications about the roles of the individual gene products or groups of proteases.

  2. The Comparison of Anti-felting Effect of Various Proteases on Wool%多种蛋白酶对羊毛防毡缩整理效果的比较

    Institute of Scientific and Technical Information of China (English)

    王岑; 王杰; 沈加加

    2014-01-01

    Pepsase, acid proteinase, papain, bromelain and alkaline protease were used in anti-felting treatment of wool. The effects of five kinds of proteases on breaking force and felting property of wool fiber were tested and analyzed, and the reaction of bromine Allworden was analyzed. The pineapple protease whose effect was best was used in anti-felting treatment in collaboration with H2O2 and chitosan. The results show that anti-felting effect of pineapple protease is the best among the five kinds of proteases, which reduces the density of wool felting ball by 31.54%, and remains the retension rate of strength by 95.7%; the reaction of bromine Allworden verifies the de_nudation of protease on wool scale layer; the collaborative process of H2O2,Protease and chitosan can maintain high strength, at the same time, it greatly decreases the density of wool felting ball.%采用胃蛋白酶、酸性蛋白酶、木瓜蛋白酶、菠萝蛋白酶、碱性蛋白酶对羊毛进行防毡缩整理,测试并分析了5种蛋白酶对羊毛纤维断裂强力和毡缩性能的影响,以及溴Allworden反应。将其中效果最好的菠萝蛋白酶协同H 2O 2和壳聚糖进行防毡缩处理。结果表明,这5种蛋白酶中菠萝蛋白酶处理效果最佳,羊毛毡缩球密度减少31.54%,强力保留率达95.7%;溴Allworden反应验证了对羊毛鳞片层的剥蚀作用;采用H 2O 2+蛋白酶+壳聚糖的协同处理工艺可在保持羊毛纤维较高强力的情况下,大大降低毡缩球密度。

  3. Extracellular acid protease from Aspergillus niger I1: purification and ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-09-15

    Sep 15, 2009 ... Proteases with high activity and stability in acid pH range have .... at a flow rate of 25 ml/h after elution with the same buffer. Protein content (abs. at ..... Role of the hydrophobic amino acid residue in the bitterness of peptides.

  4. The HIV protease inhibitor nelfinavir inhibits Kaposi's sarcoma-associated herpesvirus replication in vitro.

    Science.gov (United States)

    Gantt, Soren; Carlsson, Jacquelyn; Ikoma, Minako; Gachelet, Eliora; Gray, Matthew; Geballe, Adam P; Corey, Lawrence; Casper, Corey; Lagunoff, Michael; Vieira, Jeffrey

    2011-06-01

    Kaposi's sarcoma (KS) is the most common HIV-associated cancer worldwide and is associated with high levels of morbidity and mortality in some regions. Antiretroviral (ARV) combination regimens have had mixed results for KS progression and resolution. Anecdotal case reports suggest that protease inhibitors (PIs) may have effects against KS that are independent of their effect on HIV infection. As such, we evaluated whether PIs or other ARVs directly inhibit replication of Kaposi's sarcoma-associated herpesvirus (KSHV), the gammaherpesvirus that causes KS. Among a broad panel of ARVs tested, only the PI nelfinavir consistently displayed potent inhibitory activity against KSHV in vitro as demonstrated by an efficient quantitative assay for infectious KSHV using a recombinant virus, rKSHV.294, which expresses the secreted alkaline phosphatase. This inhibitory activity of nelfinavir against KSHV replication was confirmed using virus derived from a second primary effusion lymphoma cell line. Nelfinavir was similarly found to inhibit in vitro replication of an alphaherpesvirus (herpes simplex virus) and a betaherpesvirus (human cytomegalovirus). No activity was observed with nelfinavir against vaccinia virus or adenovirus. Nelfinavir may provide unique benefits for the prevention or treatment of HIV-associated KS and potentially other human herpesviruses by direct inhibition of replication.

  5. Taspase1: a 'misunderstood' protease with translational cancer relevance.

    Science.gov (United States)

    Wünsch, D; Hahlbrock, A; Jung, S; Schirmeister, T; van den Boom, J; Schilling, O; Knauer, S K; Stauber, R H

    2016-06-30

    Proteolysis is not only a critical requirement for life, but the executing enzymes also play important roles in numerous pathological conditions, including cancer. Therefore, targeting proteases is clearly relevant for improving cancer patient care. However, to effectively control proteases, a profound knowledge of their mechanistic function as well as their regulation and downstream signalling in health and disease is required. The highly conserved protease Threonine Aspartase1 (Taspase1) is overexpressed in numerous liquid and solid malignancies and was characterized as a 'non-oncogene addiction' protease. Although Taspase1 was shown to cleave various regulatory proteins in humans as well as leukaemia provoking mixed lineage leukaemia fusions, our knowledge on its detailed functions and the underlying mechanisms contributing to cancer is still incomplete. Despite superficial similarity to type 2 asparaginases as well as Ntn proteases, such as the proteasome, Taspase1-related research so far gives us the picture of a unique protease exhibiting special features. Moreover, neither effective genetic nor chemical inhibitors for this enzyme are available so far, thus hampering not only to further dissect Taspase1's pathobiological functions but also precluding the assessment of its clinical impact. Based on recent insights, we here critically review the current knowledge of Taspase1's structure-function relationship and its mechanistic relevance for tumorigenesis obtained from in vitro and in vivo cancer models. We provide a comprehensive overview of tumour entities for which Taspase1 might be of predictive and therapeutic value, and present the respective experimental evidence. To stimulate progress in the field, a comprehensive overview of Taspase1 targeting approaches is presented, including coverage of Taspase1-related patents. We conclude by discussing future inhibition strategies and relevant challenges, which need to be resolved by the field.

  6. Conservation of sequence and function in fertilization of the cortical granule serine protease in echinoderms.

    Science.gov (United States)

    Oulhen, Nathalie; Xu, Dongdong; Wessel, Gary M

    2014-08-01

    Conservation of the cortical granule serine protease during fertilization in echinoderms was tested both functionally in sea stars, and computationally throughout the echinoderm phylum. We find that the inhibitor of serine protease (soybean trypsin inhibitor) effectively blocks proper transition of the sea star fertilization envelope into a protective sperm repellent, whereas inhibitors of the other main types of proteases had no effect. Scanning the transcriptomes of 15 different echinoderm ovaries revealed sequences of high conservation to the originally identified sea urchin cortical serine protease, CGSP1. These conserved sequences contained the catalytic triad necessary for enzymatic activity, and the tandemly repeated LDLr-like repeats. We conclude that the protease involved in the slow block to polyspermy is an essential and conserved element of fertilization in echinoderms, and may provide an important reagent for identification and testing of the cell surface proteins in eggs necessary for sperm binding.

  7. A novel class of cysteine protease inhibitors: solution structure of staphostatin A from Staphylococcus aureus.

    Science.gov (United States)

    Dubin, Grzegorz; Krajewski, Marcin; Popowicz, Grzegorz; Stec-Niemczyk, Justyna; Bochtler, Matthias; Potempa, Jan; Dubin, Adam; Holak, Tad A

    2003-11-25

    A series of secreted proteases are included among the virulence factors documented for Staphylococcus aureus. In light of increasing antibiotic resistance of this dangerous human pathogen, these proteases are considered as suitable targets for the development of novel therapeutic strategies. The recent discovery of staphostatins, endogenous, highly specific, staphylococcal cysteine protease inhibitors, opened a possibility for structure-based design of low molecular weight analogues. Moreover, the crystal structure of staphostatin B revealed a distinct folding pattern and an unexpected, substrate-like binding mode. The solution structure of staphostatin A reported here confirms that staphostatins constitute a novel, distinct class of cysteine protease inhibitors. In addition, the structure knowledge-based mutagenesis studies shed light on individual structural features of staphostatin A, the inhibition mechanism, and the determinants of distinct specificity of staphostatins toward their target proteases.

  8. Apoptosis Mediated by HIV Protease is Preceded by Cleavage of Bcl-2

    Science.gov (United States)

    Strack, Peter R.; West Frey, Michelle; Rizzo, Christopher J.; Cordova, Beverly; George, Henry J.; Meade, Raymond; Ho, Siew Peng; Corman, Jeanne; Tritch, Radonna; Korant, Bruce D.

    1996-09-01

    Expression of the human immunodeficiency virus type 1 (HIV) protease in cultured cells leads to apoptosis, preceded by cleavage of bcl-2, a key negative regulator of cell death. In contrast, a high level of bcl-2 protects cells in vitro and in vivo from the viral protease and prevents cell death following HIV infection of human lymphocytes, while reducing the yields of viral structural proteins, infectivity, and tumor necrosis factor α . We present a model for HIV replication in which the viral protease depletes the infected cells of bcl-2, leading to oxidative stress-dependent activation of NFkappa B, a cellular factor required for HIV transcription, and ultimately to cell death. Purified bcl-2 is cleaved by HIV protease between phenylalanine 112 and alanine 113. The results suggest a new option for HIV gene therapy; bcl-2 muteins that have noncleavable alterations surrounding the HIV protease cleavage site.

  9. Interdomain Contacts and the Stability of Serralysin Protease from Serratia marcescens.

    Directory of Open Access Journals (Sweden)

    Liang Zhang

    Full Text Available The serralysin family of bacterial metalloproteases is associated with virulence in multiple modes of infection. These extracellular proteases are members of the Repeats-in-ToXin (RTX family of toxins and virulence factors, which mediated virulence in E. coli, B. pertussis, and P. aeruginosa, as well as other animal and plant pathogens. The serralysin proteases are structurally dynamic and their folding is regulated by calcium binding to a C-terminal domain that defines the RTX family of proteins. Previous studies have suggested that interactions between N-terminal sequences and this C-terminal domain are important for the high thermal and chemical stabilities of the RTX proteases. Extending from this, stabilization of these interactions in the native structure may lead to hyperstabilization of the folded protein. To test this hypothesis, cysteine pairs were introduced into the N-terminal helix and the RTX domain and protease folding and activity were assessed. Under stringent pH and temperature conditions, the disulfide-bonded mutant showed increased protease activity and stability. This activity was dependent on the redox environment of the refolding reaction and could be blocked by selective modification of the cysteine residues before protease refolding. These data demonstrate that the thermal and chemical stability of these proteases is, in part, mediated by binding between the RTX domain and the N-terminal helix and demonstrate that stabilization of this interaction can further stabilize the active protease, leading to additional pH and thermal tolerance.

  10. Screening and molecular classification of low-temperature protease from Antarctic microorganism and its characterization

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    107 strains producing protease were screened from 260 strains of Antarctic psychrophilic bacteria, among which proteolytic activity of five strains was more than 45 U ml-1. The 16S rRNA gene sequences homology and phylogenetic analysis of five Antarctic psychrophilic bacteria showed that NJ276、 NJ5-9、NJ16-70、NJ345 belonged to the described genus Pseudoalteromonas and NJ341belonged to the genus Colwellia. The growth and the protease characteristic of four Antarctic psychrophilic bacteria had been studied, and the result showed that the optimal temperature for growth and protease-producing of four strains was about 10 ℃. Their growth and protease-producing were still high during incubating 2-5 days. The maximum proteolytic activity occurred at pH 9 for four Antarctic psychrophilic bacteria. The optimal temperature of protease action of both strains NJ276 and NJ5-9 was about 50 ℃, however, the optimal temperature of protease action of both strains NJ341 and NJ345 was about 40 ℃, and their proteolytic activity under 0 ℃ exhibited nearly 30% of the maximum activity,but their thermal stabilities were weaker. These results indicated that proteases from NJ341 and NJ345 were low-temperature proteases.

  11. Development of a protease activity assay using heat-sensitive Tus-GFP fusion protein substrates.

    Science.gov (United States)

    Askin, Samuel P; Morin, Isabelle; Schaeffer, Patrick M

    2011-08-15

    Proteases are implicated in various diseases and several have been identified as potential drug targets or biomarkers. As a result, protease activity assays that can be performed in high throughput are essential for the screening of inhibitors in drug discovery programs. Here we describe the development of a simple, general method for the characterization of protease activity and its use for inhibitor screening. GFP was genetically fused to a comparatively unstable Tus protein through an interdomain linker containing a specially designed protease site, which can be proteolyzed. When this Tus-GFP fusion protein substrate is proteolyzed it releases GFP, which remains in solution after a short heat denaturation and centrifugation step used to eliminate uncleaved Tus-GFP. Thus, the increase in GFP fluorescence is directly proportional to protease activity. We validated the protease activity assay with three different proteases, i.e., trypsin, caspase 3, and neutrophil elastase, and demonstrated that it can be used to determine protease activity and the effect of inhibitors with small sample volumes in just a few simple steps using a fluorescence plate reader.

  12. Structure and mechanism of rhomboid protease.

    Science.gov (United States)

    Ha, Ya; Akiyama, Yoshinori; Xue, Yi

    2013-05-31

    Rhomboid protease was first discovered in Drosophila. Mutation of the fly gene interfered with growth factor signaling and produced a characteristic phenotype of a pointed head skeleton. The name rhomboid has since been widely used to describe a large family of related membrane proteins that have diverse biological functions but share a common catalytic core domain composed of six membrane-spanning segments. Most rhomboid proteases cleave membrane protein substrates near the N terminus of their transmembrane domains. How these proteases function within the confines of the membrane is not completely understood. Recent progress in crystallographic analysis of the Escherichia coli rhomboid protease GlpG in complex with inhibitors has provided new insights into the catalytic mechanism of the protease and its conformational change. Improved biochemical assays have also identified a substrate sequence motif that is specifically recognized by many rhomboid proteases.

  13. Cation exchange properties of zeolites in hyper alkaline aqueous media.

    Science.gov (United States)

    Van Tendeloo, Leen; de Blochouse, Benny; Dom, Dirk; Vancluysen, Jacqueline; Snellings, Ruben; Martens, Johan A; Kirschhock, Christine E A; Maes, André; Breynaert, Eric

    2015-02-03

    Construction of multibarrier concrete based waste disposal sites and management of alkaline mine drainage water requires cation exchangers combining excellent sorption properties with a high stability and predictable performance in hyper alkaline media. Though highly selective organic cation exchange resins have been developed for most pollutants, they can serve as a growth medium for bacterial proliferation, impairing their long-term stability and introducing unpredictable parameters into the evolution of the system. Zeolites represent a family of inorganic cation exchangers, which naturally occur in hyper alkaline conditions and cannot serve as an electron donor or carbon source for microbial proliferation. Despite their successful application as industrial cation exchangers under near neutral conditions, their performance in hyper alkaline, saline water remains highly undocumented. Using Cs(+) as a benchmark element, this study aims to assess the long-term cation exchange performance of zeolites in concrete derived aqueous solutions. Comparison of their exchange properties in alkaline media with data obtained in near neutral solutions demonstrated that the cation exchange selectivity remains unaffected by the increased hydroxyl concentration; the cation exchange capacity did however show an unexpected increase in hyper alkaline media.

  14. Recombinant expression and functional analysis of proteases from Streptococcus pneumoniae, Bacillus anthracis, and Yersinia pestis

    Directory of Open Access Journals (Sweden)

    Pieper Rembert

    2011-05-01

    Full Text Available Abstract Background Uncharacterized proteases naturally expressed by bacterial pathogens represents important topic in infectious disease research, because these enzymes may have critical roles in pathogenicity and cell physiology. It has been observed that cloning, expression and purification of proteases often fail due to their catalytic functions which, in turn, cause toxicity in the E. coli heterologous host. Results In order to address this problem systematically, a modified pipeline of our high-throughput protein expression and purification platform was developed. This included the use of a specific E. coli strain, BL21(DE3 pLysS to tightly control the expression of recombinant proteins and various expression vectors encoding fusion proteins to enhance recombinant protein solubility. Proteases fused to large fusion protein domains, maltosebinding protein (MBP, SP-MBP which contains signal peptide at the N-terminus of MBP, disulfide oxidoreductase (DsbA and Glutathione S-transferase (GST improved expression and solubility of proteases. Overall, 86.1% of selected protease genes including hypothetical proteins were expressed and purified using a combination of five different expression vectors. To detect novel proteolytic activities, zymography and fluorescence-based assays were performed and the protease activities of more than 46% of purified proteases and 40% of hypothetical proteins that were predicted to be proteases were confirmed. Conclusions Multiple expression vectors, employing distinct fusion tags in a high throughput pipeline increased overall success rates in expression, solubility and purification of proteases. The combinatorial functional analysis of the purified proteases using fluorescence assays and zymography confirmed their function.

  15. Proteolytic crosstalk in multi-protease networks

    Science.gov (United States)

    Ogle, Curtis T.; Mather, William H.

    2016-04-01

    Processive proteases, such as ClpXP in E. coli, are conserved enzyme assemblies that can recognize and rapidly degrade proteins. These proteases are used for a number of purposes, including degrading mistranslated proteins and controlling cellular stress response. However, proteolytic machinery within the cell is limited in capacity and can lead to a bottleneck in protein degradation, whereby many proteins compete (‘queue’) for proteolytic resources. Previous work has demonstrated that such queueing can lead to pronounced statistical relationships between different protein counts when proteins compete for a single common protease. However, real cells contain many different proteases, e.g. ClpXP, ClpAP, and Lon in E. coli, and it is not clear how competition between proteins for multiple classes of protease would influence the dynamics of cellular networks. In the present work, we theoretically demonstrate that a multi-protease proteolytic bottleneck can substantially couple the dynamics for both simple and complex (oscillatory) networks, even between substrates with substantially different affinities for protease. For these networks, queueing often leads to strong positive correlations between protein counts, and these correlations are strongest near the queueing theoretic point of balance. Furthermore, we find that the qualitative behavior of these networks depends on the relative size of the absolute affinity of substrate to protease compared to the cross affinity of substrate to protease, leading in certain regimes to priority queue statistics.

  16. Mass spectrometry-assisted protease substrate screening

    DEFF Research Database (Denmark)

    Schlüter, Hartmut; Rykl, Jana; Thiemann, Joachim;

    2007-01-01

    Since sequencing of the human genome was completed, more than 500 genes have been annotated as proteases. Exploring the physiological role of each protease requires the identification of their natural substrates. However, the endogenous substrates of many of the human proteases are as yet unknown....... Here we describe a new assay that addresses this problem. The assay, which easily can be automated, is based on the incubation of immobilized protein fractions, which may contain the natural substrate, with a defined protease. After concentrating the proteolytically released peptides by reversed...

  17. Concomitant production of two proteases and alpha-amylase by a novel strain of Bacillus subtilis in a microprocessor controlled bioreactor

    Directory of Open Access Journals (Sweden)

    Hamid Mukhtar

    2012-09-01

    Full Text Available We describe the simultaneous production of Bacillus subtilis based proteases and alpha amylase using a computer controlled laboratory scale 7.5 L batch bioreactor. The present strain is the first to be reported that concomitantly produces these two industrially important enzymes. The growth and sporulation of Bacillus subtilis was monitored and maximum production of alkaline protease and alpha amylase was found to coincide with maximum sporulation. Two types of proteases were detected in the fermentation broth; a neutral and an alkaline protease most active in a pH range of 7.0-8.0 and 8.0-10, respectively. Maximum production of proteases was observed at an incubation temperature of 37ºC while that of alpha amylase was observed at 40ºC. The optimum aeration and agitation levels for protease production were 0.6 L/L/min and 200rpm, respectively, and for alpha amylase were 0.6 L/L/min and 150 rpm. The kinetic parameters Yp/x and qp were also found to be significant at the given fermentation conditions.

  18. Alkaline phosphatase of Physarum polycephalum is insoluble.

    Science.gov (United States)

    Furuhashi, Kiyoshi

    2008-02-01

    The plasmodia of Physarum polycephalum grow as multinucleated cells in the presence of sufficient humidity and nutriment. Under non-illuminating conditions, stresses such as low temperature or high concentrations of salts transform the plasmodia into spherules whereas dehydration induces sclerotization. Some phosphatases including protein phosphatase and acid phosphatase have been purified from the plasmodia, but alkaline phosphatase remains to be elucidated. Phosphatase of the plasmodia, spherules and sclerotia was visualized by electrophoresis gel-staining assay using 5-bromo-4-chloro-3-indolyl phosphate. Insoluble fractions of the sclerotia were abundant in phosphatase activity. The phosphatase which was extracted by nonionic detergent was subjected to column chromatography and preparative electrophoresis. Purified phosphatase showed the highest activity at pH 8.8, indicating that this enzyme belongs to alkaline phosphatase. The apparent molecular mass from sodium dodecyl sulfate-polyacrylamide gel electrophoresis under non-reducing condition was estimated to be 100 kDa whereas that under reducing was 105 kDa. An amount of 1% sodium dodecyl sulfate or 0.5 M NaCl had no effects on the activity although the phosphatase showed heat instability, Mg(2+)-dependency and sensitivity to 2-glycerophosphate or NaF. The extracting conditions and enzymatic properties suggest that this alkaline phosphatase which is in a membrane-bound form plays important roles in phosphate metabolism.

  19. Impact of protease activity of yeasts on wine fermentation and formation of volatile and non-volatile metabolites

    OpenAIRE

    Chomsri, Ni-orn

    2008-01-01

    Fifty yeast strains previously isolated from grapes and wines were investigated for extracellular protease activity. Saccharomyces yeasts showed no detectable protease activity, while most of the non-Saccharomyces yeasts exhibited proteolytic activity. Nine of the fifty yeast strains were cultivated in grape juice and were analysed for the production of extracellular proteases. The proteolytic activity of Metschnikowia pulcherrima (M 004) was as high as that of Rhodotolura sp. (R3). Very low ...

  20. Middle Jurassic MORB-type gabbro, high-Mg diorite, calc-alkaline diorite and granodiorite in the Ando area, central Tibet: Evidence for a slab roll-back of the Bangong-Nujiang Ocean

    Science.gov (United States)

    Yan, Haoyu; Long, Xiaoping; Wang, Xuan-Ce; Li, Jie; Wang, Qiang; Yuan, Chao; Sun, Min

    2016-11-01

    Mesozoic intrusions, including MORB-type gabbros, high-Mg diorites, calc-alkaline diorites and granodiorites, were exposed in the Ando microcontinent that is bounded between the Qiangtang and Lhasa terranes. Discoveries of these Mesozoic intrusions have provided new petrogenetic constraints on our understanding of Bangong-Nujiang ocean evolution. Zircon U-Pb dating shows that these intrusions formed in the early-middle Jurassic (174-177 Ma). The gabbros have relatively flat REE distribution patterns, which is analogous to the geochemical features of MORB. Their positive εNd(t) values (εNd(t) = 4.4-5.5) are consistent with those of ophiolites along the Bangong-Nujiang suture zone. These gabbros are also characterized by enrichments of fluid-sensitive elements and negative to positive Nb anomalies, indicative of the influence of subduction-related compositions in their mantle source. These features suggest that the gabbros were most likely originated from asthenosphere-derived melts metasomatized by enriched lithospheric mantle during the upwelling. The high-Mg diorites are characterized by typical features of high compatible elements (MgO = 8.3-10.24 wt%, Cr = 400-547 ppm, Ni = 120-152 ppm), high Mg# (70-74) and low Sr/Y ratios. Their high initial 87Sr/86Sr isotopic ratios and negative εNd(t) values (- 10.5 to - 10.8), together with their sanukitic characteristics, imply that the high-Mg diorites were probably produced by partial melting of mantle peridotites metasomatized by slab-derived melts and aqueous fluids. The calc-alkaline diorites have relatively high MgO (4.04-5.50 wt%), Cr, Ni contents and Mg# (56-59), as well as high (86Sr/87Sr)i ratios and negative εNd(t) values (- 7.5 to - 7.3), suggesting that they were most likely formed by partial melting of the Ando basement rocks with significant input of mantle components. The granodiorites are peraluminous and have higher (86Sr/87Sr)i ratios and more negative εNd(t) values (- 10.6 to - 10.8), similar to

  1. Three monoclonal antibodies against the serpin protease nexin-1 prevent protease translocation

    DEFF Research Database (Denmark)

    Kousted, Tina Mostrup; Skjoedt, K; Petersen, S V

    2013-01-01

    , conversion to an inactive conformation or induction of serpin substrate behaviour. Until now, no inhibitory antibodies against PN-1 have been thoroughly characterised. Here we report the development of three monoclonal antibodies binding specifically and with high affinity to human PN-1. The antibodies all...... the loop connecting α-helix F with β-strand 3A and the loop connecting α-helix A with β-strand 1B. We conclude that antibody binding causes a direct blockage of the final critical step of protease translocation, resulting in abortive inhibition and premature release of reactive centre cleaved PN-1...

  2. Different contributions of HtrA protease and chaperone activities to Campylobacter jejuni stress tolerance and physiology

    DEFF Research Database (Denmark)

    Bæk, Kristoffer Torbjørn; Vegge, Christina Skovgaard; Skórko-Glonek, Joanna;

    2011-01-01

    The microaerophilic bacterium Campylobacter jejuni is the most common cause of bacterial food-borne infections in the developed world. Tolerance to environmental stress relies on proteases and chaperones in the cell envelope such as HtrA and SurA. HtrA displays both chaperone and protease activity......, but little is known about how each of these activities contributes to stress tolerance in bacteria. In vitro experiments showed temperature dependent protease and chaperone activities of C. jejuni HtrA. A C. jejuni mutant lacking only the protease activity of HtrA was used to show that the HtrA chaperone...... activity is sufficient for growth at high temperature or oxidative stress, whereas the HtrA protease activity is only essential at conditions close to the growth limit for C. jejuni. However, the protease activity was required to prevent induction of the cytoplasmic heat-shock response even at optimal...

  3. Characterization and inhibition of norovirus proteases of genogroups I and II using a fluorescence resonance energy transfer assay

    Energy Technology Data Exchange (ETDEWEB)

    Chang, Kyeong-Ok [Department of Diagnostic Medicine and Pathobiology, College of Veterinary Medicine, Kansas State University, KS 66506 (United States); Takahashi, Daisuke; Prakash, Om [Department of Biochemistry, Kansas State University, Manhattan, KS 66506 (United States); Kim, Yunjeong, E-mail: ykim@vet.ksu.edu [Department of Diagnostic Medicine and Pathobiology, College of Veterinary Medicine, Kansas State University, KS 66506 (United States)

    2012-02-20

    Noroviruses are the major cause of food- or water-borne gastroenteritis outbreaks in humans. The norovirus protease that cleaves a large viral polyprotein to nonstructural proteins is essential for virus replication and an attractive target for antiviral drug development. Noroviruses show high genetic diversity with at least five genogroups, GI-GV, of which GI and GII are responsible for the majority of norovirus infections in humans. We cloned and expressed proteases of Norwalk virus (GI) and MD145 virus (GII) and characterized the enzymatic activities with fluorescence resonance energy transfer substrates. We demonstrated that the GI and GII proteases cleaved the substrates derived from the naturally occurring cleavage site in the open reading frame (ORF) 1 of G1 norovirus with similar efficiency, and that enzymatic activity of both proteases was inhibited by commercial protease inhibitors including chymostatin. The interaction of chymostatin to Norwalk virus protease was validated by nuclear magnetic resonance (NMR) spectroscopy.

  4. Searching for halo-alkalophilic proteases maintaining stability and activity in hydrophilic aprotic solvents as biocatalysts in carbohydrate chemistry

    DEFF Research Database (Denmark)

    Pedersen, Lars Haastrup; Mørkholt, Camilla Kær; Nielsen, Carsten Bue

    2012-01-01

    BIOCAT2012, Hamburg University of Technology Book of Abstracts, p70 ISBN 987-3-941492 L12) Searching for halo-alkalophilic proteases maintaining stability and activity in hydrophilic aprotic solvents as biocatalysts in carbohydrate chemistry. Lars Haastrup Pedersen, Camilla Kær Mørkholt, Carsten...... Bue Nielsen Department of Biotechnology, Chemistry and Environmental Engineering Aalborg University Denmark Halo-alkalophilic proteases have proven relatively stable maintaining activity in hydrophilic aprotic solvents sufficiently to be useful as biocatalysts for regiospecific acylation of sucrose...... and other carbohydrates (1, 2). Therefore, we are searching for new bacterial halo-alkaline proteases from extreme environments in Denmark. So far we have identified a number of interesting isolates showing proteolytic activity at pH 7 and 10. Whole genome Illumina Hiseq 2000 amplicon sequencing, de novo...

  5. Factor VII activating protease. Single nucleotide polymorphisms light the way.

    Science.gov (United States)

    Kanse, S M; Etscheid, M

    2011-08-01

    Factor VII activating protease (FSAP) is a circulating serine protease with high homology to fibrinolytic enzymes. A role in the regulation of coagulation and fibrinolysis is suspected based on in vitro studies demonstrating activation of FVII or pro-urokinase plasminogen activator (uPA). However, considering the paucity of any studies in animal models or any correlative studies in humans the role of FSAP in haemostasis remains unclear. In relation to vascular remodeling processes or inflammation it has been convincingly shown that FSAP interacts with growth factors as well as protease activated receptors (PAR). Against this sparse background there are a plethora of studies which have investigated the linkage of single nucleotide polymorphisms (SNP) in the FSAP gene (HABP2) to various diseases. The G534E SNP of FSAP is associated with a low proteolytic activity due to an amino acid exchange in the protease domain. This and other SNPs have been linked to carotid stenosis, stroke as well as thrombosis in the elderly and plaque calcification. These SNP analyses indicate an important role for FSAP in the regulation of the haemostasis system as well as fibroproliferative inflammatory processes.

  6. 2nd Generation Alkaline Electrolysis

    DEFF Research Database (Denmark)

    Yde, Lars; Kjartansdóttir, Cecilia Kristin; Allebrod, Frank;

    This report provides the results of the 2nd Generation Alkaline Electrolysis project which was initiated in 2008. The project has been conducted from 2009-2012 by a consortium comprising Århus University Business and Social Science – Centre for Energy Technologies (CET (former HIRC)), Technical...

  7. 蛋白酶在乙醇溶液中的性质及构象%The Properties and Conformation of Proteases in Ethanol Solution

    Institute of Scientific and Technical Information of China (English)

    刘玉芳; 王淼

    2011-01-01

    对碱性蛋白酶和木瓜蛋白酶在乙醇溶液中的性质和构象进行了研究。研究结果表明,在乙醇溶液中,碱性蛋白酶和木瓜蛋白酶催化反应的最适温度有不同程度下降,下降幅度为5-10℃;它们的最适pH与在缓冲液体系中相差不大。2种蛋白酶在醇溶液中的稳定性随着乙醇浓度的升高而下降,特别是当乙醇浓度上升至60%,2种蛋白酶的稳定性均迅速下降。在相同的乙醇浓度下,木瓜蛋白酶的稳定性比碱性蛋白酶高。当乙醇浓度为60%时,木瓜蛋白酶的半衰期是碱性蛋白酶的21倍。圆二色谱研究表明,在乙醇溶液中碱性蛋白酶和木瓜蛋白酶的Z-级结构均发生了明显变化,荧光光谱表明,在乙醇溶液中2种蛋白酶的荧光强度均有明显减弱,表明它们的三级结构均发生明显变化。%The properties and conformation of alkaline protease and papain in ethanol solutions were researched. The results showed that the optimum temperature of alkaline protease and papain in ethanol solutions were reduced with the increase of ethanol concentration. The range of temperature descent was 5 - 10℃. The optimum pH in ethanol solutions had little difference comparing with buffer solution. With the increase of ethanol concentration, the stability of alkaline protease and papain were affected. Especially, when the concentration of ethanol solutions was up to 60% , the stability of alkaline protease and papain decreased rapidly. The stability of papain was higher than alkaline protease in the same ethanol concentration and the half-life of papain was 21 times of alkaline protease in 60% etha- nol. The Circular dichroism spectra of the protease in ethanol solutions showed that the secondary structure of alkaline protease and papain were altered seriously. The Fluorescence spectra of the protease ethanol solution indicated that the fluorescence intensity of alkaline protease and papain in ethanol

  8. A cyclic peptidic serine protease inhibitor

    DEFF Research Database (Denmark)

    Zhao, Baoyu; Xu, Peng; Jiang, Longguang;

    2014-01-01

    Peptides are attracting increasing interest as protease inhibitors. Here, we demonstrate a new inhibitory mechanism and a new type of exosite interactions for a phage-displayed peptide library-derived competitive inhibitor, mupain-1 (CPAYSRYLDC), of the serine protease murine urokinase...

  9. Immobilization to prevent enzyme incompatibility with proteases

    NARCIS (Netherlands)

    Vossenberg, P.; Beeftink, H.H.; Cohen Stuart, M.A.; Tramper, J.

    2011-01-01

    Enzyme incompatibility is a problem in multi-enzyme processes that involve a non-specific protease, such as Alcalase. An example is the one-pot enzymatic synthesis of peptides catalyzed by a lipase and a protease. The incompatibility between lipase B from Candida antarctica (CalB) and Alcalase was

  10. Short chain aliphatic acid anions in oil field waters and their contribution to the measured alkalinity

    Science.gov (United States)

    Willey, L.M.; Kharaka, Y.K.; Presser, T.S.; Rapp, J.B.; Barnes, I.

    1975-01-01

    High alkalinity values found in some formation waters from Kettleman North Dome oil field are due chiefly to acetate and propionate ions, with some contribution from higher molecular weight organic acid ions. Some of these waters contain no detectable bicarbonate alkalinity. For waters such as these, high supersaturation with respect to calcite will be incorrectly indicated by thermodynamic calculations based upon carbonate concentrations inferred from traditional alkalinity measurements. ?? 1975.

  11. Evolutionary optimization of peptide substrates for proteases that exhibit rapid hydrolysis kinetics.

    Science.gov (United States)

    Boulware, Kevin T; Jabaiah, Abeer; Daugherty, Patrick S

    2010-06-15

    Protease cleavage site recognition motifs can be identified using protease substrate discovery methodologies, but typically exhibit non-optimal specificity and activity. To enable evolutionary optimization of substrate cleavage kinetics, a two-color cellular library of peptide substrates (CLiPS) methodology was developed. Two-color CLiPS was applied to identify peptide substrates for the tobacco etch virus (TEV) protease from a random pentapeptide library, which were then optimized by screening of a focused, extended substrate library. Quantitative library screening yielded seven amino acid substrates exhibiting rapid hydrolysis by TEV protease and high sequence similarity to the native seven-amino-acid substrate, with a strong consensus of EXLYPhiQG. Comparison of hydrolysis rates for a family of closely related substrates indicates that the native seven-residue TEV substrate co-evolved with TEV protease to facilitate highly efficient hydrolysis. Consensus motifs revealed by screening enabled database identification of a family of related, putative viral protease substrates. More generally, our results suggest that substrate evolution using CLiPS may be useful for optimizing substrate selectivity and activity to enable the design of more effective protease activity probes, molecular imaging agents, and prodrugs.

  12. Acid transformation of bauxite residue: Conversion of its alkaline characteristics.

    Science.gov (United States)

    Kong, Xiangfeng; Li, Meng; Xue, Shengguo; Hartley, William; Chen, Chengrong; Wu, Chuan; Li, Xiaofei; Li, Yiwei

    2017-02-15

    Bauxite residue (BR) is a highly alkaline solid hazardous waste produced from bauxite processing for alumina production. Alkaline transformation appears to reduce the environmental risk of bauxite residue disposal areas (BRDAs) whilst potentially providing opportunities for the sustainable reuse and on-going management of BR. Mineral acids, a novel citric acid and a hybrid combination of acid-gypsum treatments were investigated for their potential to reduce residue pH and total alkalinity and transform the alkaline mineral phase. XRD results revealed that with the exception of andradite, the primary alkaline solid phases of cancrinite, grossular and calcite were transformed into discriminative products based on the transformation used. Supernatants separated from BR and transformed bauxite residue (TBR) displayed distinct changes in soluble Na, Ca and Al, and a reduction in pH and total alkalinity. SEM images suggest that mineral acid transformations promote macro-aggregate formation, and the positive promotion of citric acid, confirming the removal or reduction in soluble and exchangeable Na. NEXAFS analysis of Na K-edge revealed that the chemical speciation of Na in TBRs was consistent with BR. Three acid treatments and gypsum combination had no effect on Na speciation, which affects the distribution of Na revealed by sodium STXM imaging. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. Purification, primary structures and evolution of coagulant proteases from Deinagkistrodon actus venom.

    Science.gov (United States)

    Nikandrov, Nikolai N; Deshimaru, Masanobu; Tani, Ayako; Chijiwa, Takahito; Shibata, Hiroki; Chang, Chang-Chun; Fukumaki, Yasuyuki; Ito, Tatsumi; Ohno, Motonori

    2005-12-15

    Deinagkistrodon (formerly Agkistrodon) actus (Taiwan) snake venom was found to contain at least seven closely related coagulant proteases. One of them, named actibin, was purified to homogeneity by means of four chromatographic steps. Actibin acted on fibrinogen to form fibrin clots with extremely high specific activity of 1,630 NIH units/mg and preferentially released fibrinopeptide A. Actibin was an acidic glycoprotein (pI 3.4) with molecular weight of 41,000, which was reduced to 28,800 after deglycosylation with N-glycanase. The k(cat)/K(m) values of actibin for hydrolysis of tosyl-l-arginine methyl ester and benzoyl-l-arginine p-nitroanilide were one-third to a half those for thrombin, reflecting a high potency of actibin in fibrinogen clotting. The amidase activities of actibin and its family proteases were inhibited by 3,4-dichloroisocoumarin, a serine protease inhibitor, indicating that actibin and its family proteases are serine proteases. Four cDNAs, named DaP1 and DaP7-DaP9, encoding D. actus coagulant proteases were cloned. All cDNAs contain an open reading frame of 780 bp coding for 260 amino acid residues, including a signal peptide of 24 amino acid residues. Their amino acid sequences predicted are highly homologous to one another with one to five amino acid substitutions. When four D. actus protease cDNAs were compared with the cDNAs coding for Trimeresurus flavoviridis and T. gramineus venom serine proteases, accelerated evolution was clearly observed. Similarity of the nucleotide sequences of four D. actus protease cDNAs with no synonymous and one to five nonsynonymous substitutions seems not to be in direct conformity with accelerated evolution. This possibly suggests that they have evolved to a similar direction to enhance their clotting activity rather than to produce other physiological activities.

  14. Protease-degradable electrospun fibrous hydrogels

    Science.gov (United States)

    Wade, Ryan J.; Bassin, Ethan J.; Rodell, Christopher B.; Burdick, Jason A.

    2015-03-01

    Electrospun nanofibres are promising in biomedical applications to replicate features of the natural extracellular matrix (ECM). However, nearly all electrospun scaffolds are either non-degradable or degrade hydrolytically, whereas natural ECM degrades proteolytically, often through matrix metalloproteinases. Here we synthesize reactive macromers that contain protease-cleavable and fluorescent peptides and are able to form both isotropic hydrogels and electrospun fibrous hydrogels through a photoinitiated polymerization. These biomimetic scaffolds are susceptible to protease-mediated cleavage in vitro in a protease dose-dependent manner and in vivo in a subcutaneous mouse model using transdermal fluorescent imaging to monitor degradation. Importantly, materials containing an alternate and non-protease-cleavable peptide sequence are stable in both in vitro and in vivo settings. To illustrate the specificity in degradation, scaffolds with mixed fibre populations support selective fibre degradation based on individual fibre degradability. Overall, this represents a novel biomimetic approach to generate protease-sensitive fibrous scaffolds for biomedical applications.

  15. Inhibition of human lymphocyte proliferation and cleavage of interleukin-2 by Pseudomonas aeruginosa proteases

    DEFF Research Database (Denmark)

    Theander, T G; Kharazmi, A; Pedersen, B K

    1988-01-01

    This study was undertaken to determine the effect of Pseudomonas aeruginosa alkaline protease (AP) and elastase (ELA) on human lymphocyte function. AP at 50 micrograms/ml and ELA at 12 micrograms/ml caused a 50% inhibition of phytohemagglutinin-induced proliferation. There was no difference......, the inhibition was partly reversed. ELA at 10 micrograms/ml cleaved IL-2, as judged by size chromatography of a reaction mixture containing 125I-labeled IL-2 and the proteases. The ELA-digested IL-2 exhibited a reduced binding capacity to IL-2 receptors on the lymphocytes. Furthermore, treatment...... of phytohemagglutinin-stimulated lymphocytes with AP and ELA resulted in inhibition of binding of intact IL-2 to IL-2 receptors on the stimulated lymphocytes. These results indicated that P. aeruginosa-derived enzymes are able to interfere with human lymphocyte function in vitro and that this effect might be due...

  16. Alkaline Rocks and Geodynamics

    OpenAIRE

    BONIN, Bernard

    1998-01-01

    Origin of A-type alkali feldspar granites is currently the subject of a world-wide debate. Contrasting hypotheses have been proposed, which range from an entirely crustal origin to an almost complete mantle derivation. A-type alkali feldspar granites belong to either unimodal granite (rhyolite)-dominated association, or bimodal gabbro (basalt)-granite (rhyolite) suite. It is argued that (i) the ultimate mantle origin of basic to intermediate rocks is beyond doubt, (ii) highly evolved felsi...

  17. A role in immunity for Arabidopsis cysteine protease RD21, the ortholog of the tomato immune protease C14.

    Directory of Open Access Journals (Sweden)

    Takayuki Shindo

    Full Text Available Secreted papain-like Cys proteases are important players in plant immunity. We previously reported that the C14 protease of tomato is targeted by cystatin-like EPIC proteins that are secreted by the oomycete pathogen Phytophthora infestans (Pinf during infection. C14 has been under diversifying selection in wild potato species coevolving with Pinf and reduced C14 levels result in enhanced susceptibility for Pinf. Here, we investigated the role C14-EPIC-like interactions in the natural pathosystem of Arabidopsis with the oomycete pathogen Hyaloperonospora arabidopsidis (Hpa. In contrast to the Pinf-solanaceae pathosystem, the C14 orthologous protease of Arabidopsis, RD21, does not evolve under diversifying selection in Arabidopsis, and rd21 null mutants do not show phenotypes upon compatible and incompatible Hpa interactions, despite the evident lack of a major leaf protease. Hpa isolates express highly conserved EPIC-like proteins during infections, but it is unknown if these HpaEPICs can inhibit RD21 and one of these HpaEPICs even lacks the canonical cystatin motifs. The rd21 mutants are unaffected in compatible and incompatible interactions with Pseudomonas syringae pv. tomato, but are significantly more susceptible for the necrotrophic fungal pathogen Botrytis cinerea, demonstrating that RD21 provides immunity to a necrotrophic pathogen.

  18. Isozymes of bovine intestinal alkaline phosphatase. Characterization and functional studies

    Energy Technology Data Exchange (ETDEWEB)

    Besman, M.J.A.

    1986-01-01

    The membrane-associated alkaline phosphatases of calf and adult bovine small intestines have been isolated to homogeneity by a novel method developed to purify large quantities of enzyme. Chromatofocusing revealed the existence of two isozymes in calf tissue while only one form was present in the adult. The three amphiphilic metallo protein dimers were characterized as to total amino acid and carbohydrate content, zinc stoichiometries and mode of carbohydrate linkage. The molecular relationship between the three enzymes was defined by tryptic peptide HPLC-mapping and N-terminal sequencing, and demonstrated the existence of two calf isozymes of unique primary sequence, only one of which is expressed in the adult animal. In the presence of protease inhibitors, two new, higher M/sub r/ species (66,000 and 62,000 daltons vs 60,000 daltons) of adult bovine alkaline phosphatase were demonstrated by electrophoresis of /sup 32/P/sub i/-labeled tissue, probing gels by autoradiography and Western blotting. The in vivo enzyme was isolated using a modified, rapid procedure; the two higher M/sub r/ species copurified.

  19. Identification of novel drug scaffolds for inhibition of SARS-CoV 3-Chymotrypsin-like protease using virtual and high-throughput screenings.

    Science.gov (United States)

    Lee, Hyun; Mittal, Anuradha; Patel, Kavankumar; Gatuz, Joseph L; Truong, Lena; Torres, Jaime; Mulhearn, Debbie C; Johnson, Michael E

    2014-01-01

    We have used a combination of virtual screening (VS) and high-throughput screening (HTS) techniques to identify novel, non-peptidic small molecule inhibitors against human SARS-CoV 3CLpro. A structure-based VS approach integrating docking and pharmacophore based methods was employed to computationally screen 621,000 compounds from the ZINC library. The screening protocol was validated using known 3CLpro inhibitors and was optimized for speed, improved selectivity, and for accommodating receptor flexibility. Subsequently, a fluorescence-based enzymatic HTS assay was developed and optimized to experimentally screen approximately 41,000 compounds from four structurally diverse libraries chosen mainly based on the VS results. False positives from initial HTS hits were eliminated by a secondary orthogonal binding analysis using surface plasmon resonance (SPR). The campaign identified a reversible small molecule inhibitor exhibiting mixed-type inhibition with a K(i) value of 11.1 μM. Together, these results validate our protocols as suitable approaches to screen virtual and chemical libraries, and the newly identified compound reported in our study represents a promising structural scaffold to pursue for further SARS-CoV 3CLpro inhibitor development. Copyright © 2013. Published by Elsevier Ltd.

  20. Synthesis and extended activity of triazole-containing macrocyclic protease inhibitors

    DEFF Research Database (Denmark)

    Pehere, A.D.; Pietsch, M.; Gütschow, M.;

    2013-01-01

    Peptide-derived protease inhibitors are an important class of compounds with the potential to treat a wide range of diseases. Herein, we describe the synthesis of a series of triazole- containing macrocyclic protease inhibitors pre-organized into a b-strand conformation and an evaluation...... of their activity against a panel of proteases. Acyclic azidoalkyne-based aldehydes are also evaluated for comparison. The macrocyclic peptidomimetics showed considerable activity towards calpain II, cathepsin L and S, and the 20S proteasome chymotrypsin-like activity. Some of the first examples of highly potent...

  1. Safety of protease inhibitors in HIV-infected pregnant women

    Directory of Open Access Journals (Sweden)

    Chougrani I

    2013-09-01

    Full Text Available Imène Chougrani,1 Dominique Luton,1,2 Sophie Matheron,3 Laurent Mandelbrot,2,4 Elie Azria1,2 1Department of Obstetrics and Gynecology, Bichat Claude Bernard Hospital, Paris Diderot University, Paris, 2Departement Hospitalo-Universitaire "Risk and Pregnancy", Paris, 3Department of Infectious and Tropical Diseases, Bichat Claude Bernard Hospital, Paris Diderot University, Paris, 4Department of Obstetrics and Gynecology, Louis Mourier Hospital, Paris Diderot University, Colombes, France Abstract: The dire conditions of the human immunodeficiency virus/acquired immune deficiency syndrome epidemic and the immense benefits of antiretroviral prophylaxis in prevention of mother-to-child transmission far outweigh the potential for adverse effects and undeniably justify the rapid and widespread use of this therapy, despite incomplete safety data. Highly active antiretroviral therapy has now become standard care, and more than half the validated regimens include protease inhibitors. This paper reviews current knowledge of the safety of these drugs during pregnancy, in terms of maternal and fetal outcomes. Transfer of protease inhibitors across the placenta is known to be minimal, and current data about birth defects and fetal malignancies are reassuring. Maternal liver function and glucose metabolism should be monitored in women treated with protease inhibitor-based regimens, but concerns about the development of maternal resistance, should treatment be discontinued, have been shown to be groundless. Neonates should be screened for hematologic abnormalities, although these are rarely severe or permanent and are not usually related to the protease inhibitor component of the antiretroviral combination. Current findings concerning pre-eclampsia and growth restriction are discordant, and further research is needed to address the question of placental vascular complications. The increased risk of preterm birth attributed to protease inhibitors should be

  2. Structural Mechanisms of Inactivation in Scabies Mite Serine Protease Paralogues

    Energy Technology Data Exchange (ETDEWEB)

    Fischer, Katja; Langendorf, Christopher G.; Irving, James A.; Reynolds, Simone; Willis, Charlene; Beckham, Simone; Law, Ruby H.P.; Yang, Sundy; Bashtannyk-Puhalovich, Tanya A.; McGowan, Sheena; Whisstock, James C.; Pike, Robert N.; Kemp, David J.; Buckle, Ashley M.; (Monash); (Queensland Inst. of Med. Rsrch.)

    2009-08-07

    The scabies mite (Sarcoptes scabiei) is a parasite responsible for major morbidity in disadvantaged communities and immuno-compromised patients worldwide. In addition to the physical discomfort caused by the disease, scabies infestations facilitate infection by Streptococcal species via skin lesions, resulting in a high prevalence of rheumatic fever/heart disease in affected communities. The scabies mite produces 33 proteins that are closely related to those in the dust mite group 3 allergen and belong to the S1-like protease family (chymotrypsin-like). However, all but one of these molecules contain mutations in the conserved active-site catalytic triad that are predicted to render them catalytically inactive. These molecules are thus termed scabies mite inactivated protease paralogues (SMIPPs). The precise function of SMIPPs is unclear; however, it has been suggested that these proteins might function by binding and protecting target substrates from cleavage by host immune proteases, thus preventing the host from mounting an effective immune challenge. In order to begin to understand the structural basis for SMIPP function, we solved the crystal structures of SMIPP-S-I1 and SMIPP-S-D1 at 1.85 {angstrom} and 2.0 {angstrom} resolution, respectively. Both structures adopt the characteristic serine protease fold, albeit with large structural variations over much of the molecule. In both structures, mutations in the catalytic triad together with occlusion of the S1 subsite by a conserved Tyr200 residue is predicted to block substrate ingress. Accordingly, we show that both proteases lack catalytic function. Attempts to restore function (via site-directed mutagenesis of catalytic residues as well as Tyr200) were unsuccessful. Taken together, these data suggest that SMIPPs have lost the ability to bind substrates in a classical 'canonical' fashion, and instead have evolved alternative functions in the lifecycle of the scabies mite.

  3. Mast cell proteases as pharmacological targets.

    Science.gov (United States)

    Caughey, George H

    2016-05-05

    Mast cells are rich in proteases, which are the major proteins of intracellular granules and are released with histamine and heparin by activated cells. Most of these proteases are active in the granule as well as outside of the mast cell when secreted, and can cleave targets near degranulating mast cells and in adjoining tissue compartments. Some proteases released from mast cells reach the bloodstream and may have far-reaching actions. In terms of relative amounts, the major mast cell proteases include the tryptases, chymases, cathepsin G, carboxypeptidase A3, dipeptidylpeptidase I/cathepsin C, and cathepsins L and S. Some mast cells also produce granzyme B, plasminogen activators, and matrix metalloproteinases. Tryptases and chymases are almost entirely mast cell-specific, whereas other proteases, such as cathepsins G, C, and L are expressed by a variety of inflammatory cells. Carboxypeptidase A3 expression is a property shared by basophils and mast cells. Other proteases, such as mastins, are largely basophil-specific, although human basophils are protease-deficient compared with their murine counterparts. The major classes of mast cell proteases have been targeted for development of therapeutic inhibitors. Also, a human β-tryptase has been proposed as a potential drug itself, to inactivate of snake venins. Diseases linked to mast cell proteases include allergic diseases, such as asthma, eczema, and anaphylaxis, but also include non-allergic diseases such as inflammatory bowel disease, autoimmune arthritis, atherosclerosis, aortic aneurysms, hypertension, myocardial infarction, heart failure, pulmonary hypertension and scarring diseases of lungs and other organs. In some cases, studies performed in mouse models suggest protective or homeostatic roles for specific proteases (or groups of proteases) in infections by bacteria, worms and other parasites, and even in allergic inflammation. At the same time, a clearer picture has emerged of differences in the

  4. Protease activation of the entomocidal protoxin of Bacillus thuringiensis subsp. kurstaki.

    Science.gov (United States)

    Andrews, R E; Bibilos, M M; Bulla, L A

    1985-01-01

    Two isolates of Bacillus thuringiensis subsp. kurstaki were examined which produced different levels of intracellular proteases. Although the crystals from both strains had comparable toxicity, one of the strains, LB1, had a strong polypeptide band at 68,000 molecular weight in the protein from the crystal; in the other, HD251, no such band was evident. When the intracellular proteases in both strains were measured, strain HD251 produced less than 10% of the proteolytic activity found in LB1. These proteases were primarily neutral metalloproteases, although low levels of other proteases were detected. In LB1, the synthesis of protease increased as the cells began to sporulate; however, in HD251, protease activity appeared much later in the sporulation cycle. The protease activity in strain LB1 was very high when the cells were making crystal toxin, whereas in HD251 reduced proteolytic activity was present during crystal toxin synthesis. The insecticidal toxin (molecular weight, 68,000) from both strains could be prepared by cleaving the protoxin (molecular weight, 135,000) with trypsin, followed by ion-exchange chromatography. The procedure described gave quantitative recovery of toxic activity, and approximately half of the total protein was recovered. Calculations show that these results correspond to stoichiometric conversion of protoxin to insecticidal toxin. The toxicities of whole crystals, soluble crystal protein, and purified toxin from both strains were comparable. Images PMID:3909962

  5. Engineering of TEV protease variants by yeast ER sequestration screening (YESS) of combinatorial libraries.

    Science.gov (United States)

    Yi, Li; Gebhard, Mark C; Li, Qing; Taft, Joseph M; Georgiou, George; Iverson, Brent L

    2013-04-30

    Myriad new applications of proteases would be enabled by an ability to fine-tune substrate specificity and activity. Herein we present a general strategy for engineering protease selectivity and activity by capitalizing on sequestration of the protease to be engineered within the yeast endoplasmic reticulum (ER). A substrate fusion protein composed of yeast adhesion receptor subunit Aga2, selection and counterselection substrate sequences, multiple intervening epitope tag sequences, and a C-terminal ER retention sequence is coexpressed with a protease library. Cleavage of the substrate fusion protein by the protease eliminates the ER retention sequence, facilitating transport to the yeast surface. Yeast cells that display Aga2 fusions in which only the selection substrate is cleaved are isolated by multicolor FACS with fluorescently labeled antiepitope tag antibodies. Using this system, the Tobacco Etch Virus protease (TEV-P), which strongly prefers Gln at P1 of its canonical ENLYFQ↓S substrate, was engineered to recognize selectively Glu or His at P1. Kinetic analysis indicated an overall 5,000-fold and 1,100-fold change in selectivity, respectively, for the Glu- and His-specific TEV variants, both of which retained high catalytic turnover. Human granzyme K and the hepatitis C virus protease were also shown to be amenable to this unique approach. Further, by adjusting the signaling strategy to identify phosphorylated as opposed to cleaved sequences, this unique system was shown to be compatible with the human Abelson tyrosine kinase.

  6. Optimized production of extracellular proteases by Bacillus subtilis from degraded abattoir waste

    Directory of Open Access Journals (Sweden)

    PALLAVI BADHE

    2016-04-01

    Full Text Available Proteases are ubiquitous in occurrence and are found in all living organisms. These are essential for cell growth and differentiation. The extracellular proteases are of a high commercial value and find multiple applications in various industrial sectors. The present study describes the screening of protease producing bacteria from a hitherto unexplored source i.e. degraded waste from abattoir. Three isolates were found namely yellow, white and orange coloured bacteria. Amongst them, white colored colony was found to be more suitable for protease production. The morphological, cultural, biochemical and 16S rRNA confirmed that the isolate was Bacillus subtilis. Physical and chemical parameters were optimized for maximum protease<