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Sample records for alkaline protease production

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

    African Journals Online (AJOL)

    Tuoyo Aghomotsegin

    2016-11-23

    Nov 23, 2016 ... Key words: Production, alkaline protease, Bacillus subtilis, animal wastes, enzyme activity. ... Generally, alkaline proteases are produced using submerged fermentation .... biopolymer concentrations were reported to have an influence ... adding nitrogenous compounds stimulate microorganism growth and ...

  2. Optimization of alkaline protease production and its fibrinolytic ...

    African Journals Online (AJOL)

    Optimization of alkaline protease production and its fibrinolytic activity from the ... nitrogen sources and sodium chloride concentration for protease production by the ... exploited to assist in protein degradation in various industrial processes.

  3. Microbial alkaline proteases: Optimization of production parameters and their properties

    Directory of Open Access Journals (Sweden)

    Kanupriya Miglani Sharma

    2017-06-01

    Full Text Available Proteases are hydrolytic enzymes capable of degrading proteins into small peptides and amino acids. They account for nearly 60% of the total industrial enzyme market. Proteases are extensively exploited commercially, in food, pharmaceutical, leather and detergent industry. Given their potential use, there has been renewed interest in the discovery of proteases with novel properties and a constant thrust to optimize the enzyme production. This review summarizes a fraction of the enormous reports available on various aspects of alkaline proteases. Diverse sources for isolation of alkaline protease producing microorganisms are reported. The various nutritional and environmental parameters affecting the production of alkaline proteases in submerged and solid state fermentation are described. The enzymatic and physicochemical properties of alkaline proteases from several microorganisms are discussed which can help to identify enzymes with high activity and stability over extreme pH and temperature, so that they can be developed for industrial applications.

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

    African Journals Online (AJOL)

    STORAGESEVER

    2008-05-16

    May 16, 2008 ... After 72 h incubation in a shaker incubator ... different incubation times (0 to 72 h) were investigated. Alkaline .... of alkaline protease (75%) and 24% of total protein is precipitated. ... starches and wheat flour as carbon source on protease production .... JP 395, method of making and detergent composition.

  5. Optimization of alkaline protease production from Pseudomonas ...

    African Journals Online (AJOL)

    PRECIOUS

    2009-12-15

    Dec 15, 2009 ... protease production was 37°C at pH 9, with 2% inoculum in the medium for 24 h. .... Positive. Catalase test. Positive ... The enzyme activity gradually decreases from ... Effect of temperature on protease production by Pseudomonas fluorescens. 0 .... between RNA polymerase and upstream promotes DNA.

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

    African Journals Online (AJOL)

    Among various nitrogen sources, yeast extract was found to be the best inducer of alkaline protease. Among metal salts, KNO3 and NH4Cl were found to increase protease production. The maximum enzyme production (3600 U/ml) was observed with pomegranate peels of fermentation medium in the presence of yeast ...

  7. Comparative Detection of Alkaline Protease Production in Exiguobacterium acetylicum

    International Nuclear Information System (INIS)

    Gomaa, O.M.; EI Shafey, H.M.

    2009-01-01

    Alkaline protease is one of the most important enzymes in industry, medicine, and research. In the present work, a comparative detection for alkaline protease activity was established for instant detection of enzyme activity. Eight different alkalophilic bacterial isolates were compared based on the clear zone they produced on skim milk agar. One strain gave an absolute clear zone in 16 hours and was used for alkaline protease detection. The result of Phenotypic identification using Biology Microlog 3 identified the isolate as Exiguobacterium acetylicum. The isolate under study showed slightly different characteristics from a known Exiguobacterium acetylicum strain. The isolate tolerated alkaline conditions up to ph 11, while good growth was evident at ph 7, the maximum alkaline protease activity was observed at ph 9 which reached up to 109.01 U/ml. The alkaline activity assay using alkaline protease enzyme assay were coordinating with those obtained by conductivity; there was a relevant decrease in conductivity at the maximum increase in enzyme activity, which proved the cell membrane conductivity has a close relation to alkaline protease production. This isolate has tolerated gamma radiation, the increase in dose (up to 4 Gy) gave wider clear zones in terms of diameter and this was relevant to the conductivity measurements

  8. Production of alkaline protease by Teredinobacter turnirae cells ...

    African Journals Online (AJOL)

    The conditions for immobilizing the new alkaline protease-producing bacteria strain Teredinobacter turnirae by entrapment in calcium alginate gel were investigated. The influence of alginate concentration (20, 25 and 30 g/l) and initial cell loading (ICL) on enzyme production were studied. The production of alkaline ...

  9. Hyper production of alkaline protease by mutagenized bacillus subtilis

    International Nuclear Information System (INIS)

    Qureshi, A.M.; Tanseem, F.

    2010-01-01

    The purpose of this work was to augment the alkaline protease production from Bacillus subtilis by using chemical mutagen (MMS) and UV mutagenesis. A number of mutants were isolated which produce high levels of extra cellular proteases. Analysis of culture supernatants of these mutants had shown that the total amounts of proteolysis activity were increased from 1 to 2 fold over the wild strain. Clones showing promote response were further characterized by analyzing different parameters; like of Temperature, pH substrate concentration and incubation period, to study the activity of protease enzyme. (author)

  10. Production and partial characterization of alkaline protease from bacillus subtilis mutant induced by gamma radiation

    International Nuclear Information System (INIS)

    Ibrahim, H.M.M.; Bashandy, A.S.

    2010-01-01

    Fourteen bacterial isolates belonging to B.subtilis were locally isolated from soil and screened for alkaline protease production. Only one strain, the highly potent one, was selected as alkaline protease producer and subjected to further studies to optimize its production. Alkaline protease production was maximum at 35 degree C after 72 h of incubation and at ph 10.0. molasses as a carbon source and combination of peptone and yeast extract as a nitrogen source enhanced greatly alkaline protease production. The mutant strain induced by gamma radiation showed higher alkaline protease production by 1.97 fold as compared with the parent strain. The alkaline protease enzyme was active at 40 degree C and ph 10. It was compatible with many commercial detergents and showed high stability (84 %) of its original activity with Ariel detergent. Moreover, alkaline protease enhanced the washing performance, and retained 95 % of its activity in the formulated dry powder.

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

    Directory of Open Access Journals (Sweden)

    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.

  12. Alkaline protease contributes to pyocyanin production in Pseudomonas aeruginosa.

    Science.gov (United States)

    Iiyama, Kazuhiro; Takahashi, Eigo; Lee, Jae Man; Mon, Hiroaki; Morishita, Mai; Kusakabe, Takahiro; Yasunaga-Aoki, Chisa

    2017-04-01

    The role of the alkaline protease (AprA) in pyocyanin production in Pseudomonas aeruginosa was investigated. AprA was overproduced when a plasmid carrying the aprA gene was introduced to an aprA-deletion mutant strain, EG03; thus, aprA-complemented EG03 was used as an overproducing strain. The complemented strain produced higher pyocyanin than the mutant strain in all commercially available media evaluated. Particularly, pyocyanin production was higher in the complemented than in the parental strain in brain-heart infusion and tryptic soy broths. These results suggested that protein degradation products by AprA were utilized for pyocyanin production. Protein-rich media were used in subsequent validation studies. Similar results were obtained when the basal medium was supplemented with casein or skim milk as the sole organic nitrogen source. However, gelatin failed to induce abundant pyocyanin production in the complemented strain, despite the presence of protein degradation products by AprA as assessed by SDS-PAGE. Thus, gelatin degradation products may not be suitable for pyocyanin synthesis. In conclusion, AprA could contribute to pyocyanin production in the presence of several proteins or peptides. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

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

    African Journals Online (AJOL)

    Tuoyo Aghomotsegin

    2016-11-23

    Nov 23, 2016 ... A new strain of Bacillus sp. was isolated from alkaline soil, which was able to produce extracellular alkaline ... rice and dates (Khosravi-Darani et al., 2008), protein by- products from lather ..... Pigeon pea waste as a novel ...

  14. Process optimization by response surface methodology for extracellular alkaline protease production from bacillus subtilis

    International Nuclear Information System (INIS)

    Mushtaq, Z.; Adnan, A.; Mehmood, Z.

    2014-01-01

    Three microbial cultures Bacillus subtilis DSM 1970, Bacillus subtilis GCU-8 and Bacillus licheniformis DSM 1969 were screened for protease production by casein agar plate method. Among these Bacillus subtilis GCU-8 was found to be the most potent protease producer in wide pH range (5.0 to 8.0). Fermentation conditions were optimized for the production of alkaline protease using two statistical tools: Placket Burmen Model for linear regression study and Response Surface Model for interactive effects of significant factors on production. The alkaline protease was optimally produced after 48 hours of incubation at 37 degree C in fermentation media containing equal amounts of substrates (soybean meal and wheat bran, 7.5 g), MgSO/sub 4/ 7H/sub 2/O, 0.10 g and yeast extract 0.55 g. The protease was purified to homogeneity by salt precipitation, ion-exchange chromatography and size exclusion chromatography. The homogeneity and molecular weights were checked by SDS-PAGE. The protease was 45 KDa protein, predominantly alkaline and optimally active at pH 8.0. (author)

  15. Alkaline protease production by alkaliphilic marine bacteria isolated ...

    African Journals Online (AJOL)

    The molecular mass determined using SDS-PAGE, was nearly 31.0 39 kDa. Some fundamental properties like effects of different temperatures, pH, metal ions (Ca2+, Mg2+, Cu2+, Pb3+, Mn2+ and Cd2+) and ethylene diamine tetraacetic acid (EDTA) on protease activity were also studied. Maximum activities were obtained ...

  16. 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.

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

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    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.

  18. Alkaline protease production from industrial wastes by bacillus subtilis ML-4

    International Nuclear Information System (INIS)

    Sher, M.G.; Nadeem, M.; Syed, Q.; Irfan, M.; Baig, S.

    2010-01-01

    The influence of various culture conditions on protease production by Bacillus subtilis ML-4 was studied in the presence of growth medium containing poultry feed waste (5%), K/sub 2/HPO/sub 4/ (0.3%), CaCl/sub 2/ (0.03%) and MgSO/sub 4/ (0.015%). Maximum protease production (264.25 +- 1.86 U/ml) was observed at initial pH 9 with 3% (v/v) of inoculum size after 48 h of incubation at 37 degree C. The alkaline protease was stable over a broad range of temperature (30 to 60 degree C) and pH (8 to 11). However, maximum activity (155.45 U/ml) was observed at temperature 50 degree C and pH 10. (author)

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

    African Journals Online (AJOL)

    Hacene

    2016-06-29

    Jun 29, 2016 ... The enzyme production media are optimized according to statistical methods while using two plans of ..... Coagulation. -. Nitrate reduction. + .... (i)Fructose: is a simple sugar found naturally in fruits and honey but can be ...

  20. Production of alkaline protease and larvicidal biopesticides by an ...

    African Journals Online (AJOL)

    Notably, maximum proteolytic activity was achieved with casein as a substrate followed by skim milk, gelatin and bovine serum albumin. The crude AP enzyme activity exhibited quasi-linear response with enzyme concentrations up to 0.25 mg ml-1. The isolated B. sphaericus might be employed for the economic production ...

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

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

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    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.

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

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    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.

  4. 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.

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

    NARCIS (Netherlands)

    Tjalsma, H.; Koetje, E.J.; Kiewiet, R.; Kuipers, O.P.; Kolkman, M.J.M.; Laan, J.H. van der; 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

  6. 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

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

    Science.gov (United States)

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

    2015-01-01

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

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

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    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.

  9. 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. Copyright © 2014. Published by Elsevier B.V.

  10. Immobilization of Bacillus megaterium MTCC 2444 by Ca-alginate entrapment method for enhanced alkaline protease production

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    Soma Mrudula

    2012-02-01

    Full Text Available Optimization of culture conditions and immobilization parameters for alkaline protease production was carried out by employing Bacillus megaterium MTCC2444. The partially purified enzyme was tested for its stability in the presence of oxidants, surfactants and commercial detergents. The optimum temperature, pH, incubation time and inoculum size were 55 ºC, 11, 48 h, 1 %, respectively. Calcium alginate was used as the immobilization matrix and the effects of gel concentration, bead size, age of immobilized cells, solidification period and initial biomass concentration on alkaline protease production and cell leakage were investigated. The results indicated that the immobilization was most effective with 4 % gel concentration, bead size of 3 mm, 24 h aged immobilized cells for a solidification period of 12 h at 1.5 % initial biomass concentration. The enzyme showed good stability in the presence of oxidants, surfactants and commercial detergents.

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

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    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++.

  12. Production and biochemical characterization of an alkaline protease from Aspergillus oryzae CH93.

    Science.gov (United States)

    Salihi, Ahsan; Asoodeh, Ahmad; Aliabadian, Mansour

    2017-01-01

    In this study, Aspergillus oryzae CH93 was isolated from soil sample and examined using molecular analysis. Following culture of A. oryzae CH93 under optimal enzyme production, a 47.5kDa extracellular protease was purified using ammonium sulfate precipitation and Q-Sepharose chromatography. The optimal pH 8 and temperature of 50°C obtained for the isolated protease. Sodium dodecyl sulfate (SDS), cetyltrimethyl ammonium bromide (CTAB), H 2 O 2 decreased activity, while Triton X-100 and phenylmethanesulfonyl fluoride (PMSF) had no inhibitory effect on the enzyme activity; meanwhile, 2-mercaptoethanol and ethylenediaminetetraacetic acid (EDTA) declined the protease activity. Isoamyl alcohol and acetone (30%) enhanced activity whereas 2-propanol, isopropanol and dimethyl sulfoxide (DMSO) (30%) reduced protease activity. The enzyme exhibited a half-life of 100min at its optimum temperature. Among five substrates of bovine serum albumin (BSA), N-acetyl-l-tyrosine ethyl ester monohydrate (ATEE), casein, azocasein and gelatin results showed that casein is the best substrate with V max of 0.1411±0.004μg/min and K m of 2.432±0.266μg/ml. In conclusion, the extracted protease from A. oryzae CH93 as a fungal source possessed biochemical features which could be useful in some application usages. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. Stimulatory effect of medium ingredients on alkaline protease production by bacillus licheniformis N-2 and compatibility studies with commercial detergents

    International Nuclear Information System (INIS)

    Nadeem, M.; Baig, S.; Qazi, J.I.

    2008-01-01

    Suitable concentration of ingredients of the growth medium played a vital role in production of alkaline protease by Bacillus licheniformis. Maximum enzyme activity (875.05 PU/ml) was achieved when the bacterium was grown in the medium containing glucose (1%), soybean meal (1%), K/sub 2/ HPO/sub 4/ (0.5%), MgSO/sub 4/ 7H/sub 2/O (0.05%), NaCI (0.05%), CaCI/sub 2/ 2H/sub 2/O (0.05%) at 37 degree C on 24 h incubation period with agitation of 140 rpm in shake flask cultures. More than 1% glucose decreased the enzyme production. The protease had excellent stability with wide range of Commercial detergents such as Ariel, Bonus, Bright Total, Surf Excel, Wheel and non-branded detergents, recommending its use as an effective additive in detergent formulation. (author)

  14. Isolation, characterization and optimization of culture parameters for production of an alkaline protease isolated from Aspergillus tamarii.

    Science.gov (United States)

    Anandan, Dayanandan; Marmer, William N; Dudley, Robert L

    2007-05-01

    Aspergillus tamarii expresses an extracellular alkaline protease that we show to be effective in removing hair from cattle hide. Large quantities of the enzyme will be required for the optimization of the enzymatic dehairing process so the growth conditions for maximum protease expression by A. tamarii were optimized for both solid-state culture on wheat bran and for broth culture. Optimal protease expression occurred, for both cultural media, at initial pH 9; the culture was incubated at 30 degrees C for 96 h using a 5% inoculum. The crude enzyme was isolated, purified and characterized using MALDI TOF TOF. The alkaline protease was homologous to the alkaline protease expressed by Aspergillus viridinutans.

  15. Isolation of alkaline protease from Bacillus subtilis AKRS3

    African Journals Online (AJOL)

    ashok

    2012-08-28

    Aug 28, 2012 ... production proved high protease production than the other tested ... Crude alkaline protease was most active at 55°C, pH 9 with casein as ... 13416 Afr. J. Biotechnol. ... The Gram-positive, aerobic, rod-shaped endospore-.

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

    African Journals Online (AJOL)

    User

    2011-05-09

    May 9, 2011 ... cereus and Bacillus polymyxa simultaneously with the start of sporulation phase as a ... microbial forms to inactivation by chemical or physical agents. .... alkaline pH, 9, 10 and 11 and the pH of the culture media was optimized with .... incubation temperature for alkaline protease production by Bacillus ...

  17. 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.

  18. Optimization of Nutrients and Culture Conditions for Alkaline Protease Production Using Two Endophytic Micrococci: Micrococcus aloeverae and Micrococcus yunnanensis.

    Science.gov (United States)

    Prakash, Om; Nimonkar, Yogesh; Chavadar, Mahesh S; Bharti, Nidhi; Pawar, Shrikant; Sharma, Ashutosh; Shouche, Yogesh S

    2017-06-01

    An endophytic species of Micrococcus was isolated from Aloe vera leaf (syn. Aloe barbadensis ) and screened for protease production with five other species of Micrococcus . Data indicated that endophytic Micrococcus aloeverae AE-6 MCC 2184 T and Micrococcus yunnanensis DSM 21948 T showed efficient protease production potential and secreted active protease at high salt (10%), temperature (40 °C) and in wide range of pH 8-10. Unlike M . yunnanensis DSM 21948 T , protease production by M . aloeverae AE-6 MCC 2184 T was stringently controlled by pH. Protease induction study using different group of peptides, peptide carbohydrates and peptide macronutrient combinations showed variable response with both the organisms. Result indicated that the amount of protease was not directly related to cell biomass but it depends on nature of inducible peptides. In this study we also developed a modified agar-well assay for semi-quantitative data from large number of replicates.

  19. tolerant alkaline protease from Bacillus coagulans PSB

    African Journals Online (AJOL)

    oyaide

    2013-05-22

    May 22, 2013 ... suggest the suitability of the enzyme for applications in peptide synthesis, detergent formulation and ... The cell free supernatant was recovered as crude enzyme preparation and used for further studies. Assay of protease activity. Protease activity was ... Effect of pH on growth and protease production.

  20. pH regulation of recombinant glucoamylase production in Fusarium venenatum JeRS 325, a transformant with a Fusarium oxysporum alkaline (trypsin-like) protease promoter.

    Science.gov (United States)

    Wiebe, M G; Robson, G D; Shuster, J R; Trinci, A P

    1999-08-05

    Fusarium venenatum (formerly Fusarium graminearum) JeRS 325 produces heterologous glucoamylase (GAM) under the regulation of a Fusarium oxysporum alkaline (trypsin-like) protease promoter. The glucoamylase gene was used as a reporter gene to study the effects of ammonium and pH on GAM production under the control of the alkaline protease promoter. Between pH 4.0 and 5.8, GAM production in glucose-limited chemostat cultures of JeRS 325 grown at a dilution rate of 0.10 h-1 (doubling time, 6.9 h) on (NH4)2SO4 medium increased in a linear manner with increase in pH. However, at pH 4.0 and below GAM production was almost completely repressed in glucose-limited chemostat cultures grown on (NH4)2SO4 or NaNO3 medium. Thus GAM production in JeRS 325 is regulated by culture pH, not by the nature of the nitrogen source in the medium. The difficulty of using unbuffered medium when investigating putative ammonium repression is also shown. The study demonstrates the potential for use of the alkaline protease promoter in F. graminearum for the production of recombinant proteins in a pH dependent man ner. Copyright 1999 John Wiley & Sons, Inc.

  1. 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

    Science.gov (United States)

    Haq, Ikramul

    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 byproducts tested, soybean meal at a concentration of 20 g/L gave maximum production of the enzyme, that is, 5.74  ±  0.26 U/mL from wild and 11.28  ±  0.45 U/mL from mutant strain, during submerged fermentation. Different mesh sizes (coarse, medium, and fine) of the soybean meal were also evaluated, and a finely ground soybean meal (fine mesh) was found to be the best. In addition to the defatted seed meals, their alkali extracts were also tested for the production of alkaline protease by Bacillus subtilis, but these were proved nonsignificant for enhanced production of the enzyme. The production of the enzyme was also studied in solid state fermentation, and different agro-industrial byproducts were also evaluated for enzyme production. Wheat bran partially replaced with guar meal was found as the best substrate for maximum enzyme production under solid state fermentation conditions. PMID:24294129

  2. 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

    Directory of Open Access Journals (Sweden)

    Hamid Mukhtar

    2013-01-01

    Full Text Available 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 byproducts tested, soybean meal at a concentration of 20 g/L gave maximum production of the enzyme, that is, 5.74  ±  0.26 U/mL from wild and 11.28  ±  0.45 U/mL from mutant strain, during submerged fermentation. Different mesh sizes (coarse, medium, and fine of the soybean meal were also evaluated, and a finely ground soybean meal (fine mesh was found to be the best. In addition to the defatted seed meals, their alkali extracts were also tested for the production of alkaline protease by Bacillus subtilis, but these were proved nonsignificant for enhanced production of the enzyme. The production of the enzyme was also studied in solid state fermentation, and different agro-industrial byproducts were also evaluated for enzyme production. Wheat bran partially replaced with guar meal was found as the best substrate for maximum enzyme production under solid state fermentation conditions.

  3. Purification and characterization of alkaline proteases from aspergillus terreus

    International Nuclear Information System (INIS)

    Hussain, A.; Mannan, A.; Zubair, H.; Mirza, B.

    2010-01-01

    Proteases belong to an important class of enzymes known as hydrolases and catalyze hydrolysis of proteins. They act primarily to degrade proteins that are used for energy production and as biosynthetic precursors. In the following study, protease produced from Aspergillus terreus was found to be thermo stable and included in the category of alkaline serine and metallo protease. During partial purification, presence of enzyme in 60% (NH/sub 4/)/sub 2/SO/sub 4/ indicated small molecular weight polypeptide; later purification with Sephadex G-75 fractionation yielded a single proteolytic active molecule. At final purification step, the increase in specific activity of the enzyme was 7.5 fold with 23% yield. SDS-PAGE analysis revealed that alkaline protease of Aspergillus terreus is a monomer with approximate molecular weight of 35 kDa. Optimum pH for protease activity was found in the range of 7.5-11.0 (maximum at pH 8.5), thus apparently classified as an alkaline protease. The enzyme was thermo stable towards high temperature (60 deg. C), however it denatured irreversibly at 70 deg. C showing 80% loss of activity. The maximum proteolytic activity was found at 40 deg. C. The enzyme was effectively inhibited by PMSF, EDTA and urea whereas iodoacetamide and thiourea did not result in any loss in activity while cysteine was found to be activator molecule. The study with metal ions Mg/sup +2/, Mn/sup +2/ and Fe/sup +3/ (1 mM each) showed minute stimulatory effects on enzyme activity. Co/sup +2/ and Ca/sup +2/ (1 mM) had neither excitatory nor inhibitory effect while Hg/sup +2/ and Cu/sup +2/ (1 mM) slightly reduced the enzyme activity. (author)

  4. Partial purification and characterization of alkaline proteases from ...

    African Journals Online (AJOL)

    Alkaline proteases from the digestive tract of anchovy were partially purified by ammonium sulfate fractionation, dialysis and Sephadex G-75 gel filtration. The purification fold and yield were 6.23 and 4.49%, respectively. The optimum activities of partially purified alkaline proteases were observed at 60°C and at pH 11.0.

  5. 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.

  6. A novel nonionic surfactant- and solvent-stable alkaline serine protease from Serratia sp. SYBC H with duckweed as nitrogen source: production, purification, characteristics and application.

    Science.gov (United States)

    Li, G Y; Cai, Y J; Liao, X R; Yin, J

    2011-07-01

    A novel nonionic surfactant- and hydrophilic solvent-stable alkaline serine protease was purified from the culture supernatant of Serratia sp. SYBC H with duckweed as nitrogen source. The molecular mass of the purified protease is about 59 kDa as assayed via SDS-PAGE. The protease is highly active over the pH range between 5.0 and 11.0, with the maximum activity at pH 8.0. It is also fairly active over the temperature range between 30 and 80°C, with the maximum activity at 40°C. The protease activity was substantially stimulated by Mn(2+) and Na(+) (5 mM), up to 837.9 and 134.5% at 40°C, respectively. In addition, Mn(2+) enhanced the thermostability of the protease significantly at 60°C. Over 90% of its initial activity remained even after incubating for 60 min at 40°C in 50% (v/v) hydrophilic organic solvents such as DMF, DMSO, acetone and MeOH. The protease retained 81.7, 83.6 and 76.2% of its initial activity in the presence of nonionic surfactants 20% (v/v) Tween 80, 25% (v/v) glycerol and Triton X-100, respectively. The protease is strongly inhibited by PMSF, suggesting that it is a serine protease. Washing experiments revealed that the protease has an excellent ability to remove blood stains.

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

    African Journals Online (AJOL)

    AJL

    2012-02-16

    Feb 16, 2012 ... compared with that of wild-type B. licheniformis CICIM B5102. Key word: Alkaline protease, Bacillus amyloliquefaciens, Bacillus licheniformis. INTRODUCTION. Proteases are one of the most important industrial enzyme groups, accounting for approximately 60% of the total enzyme sales (Beg et al., 2003).

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

    Directory of Open Access Journals (Sweden)

    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.

  9. Expression and Characterization of Coprothermobacter proteolyticus Alkaline Serine Protease

    Directory of Open Access Journals (Sweden)

    Tanveer Majeed

    2013-01-01

    Full Text Available 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 that the enzyme had optimal activity under alkaline conditions (pH 8–10. In addition, the enzyme had an elevated optimum temperature (60°C. The protease was also stable in the presence of many surfactants and oxidant. Thus, the C. proteolyticus protease has potential applications in industries such as the detergent market.

  10. Alkaline protease from senesced leaves of invasive weed Lantana ...

    African Journals Online (AJOL)

    STORAGESEVER

    2008-12-17

    Dec 17, 2008 ... amongst the most valuable commercial enzyme. Alkaline proteases hold a great potential for application in the detergent and leather industries (Kumar and Takagi,. 1999; Oberoi et al., 2001) due to the increasing trend to develop environmentally friendly technologies. Plants, animals and microbes are the ...

  11. An oxidant, detergent and salt stable alkaline protease from Bacillus ...

    African Journals Online (AJOL)

    A novel soil bacterium, Bacillus cereus SIU1 was earlier isolated from non-saline, slightly alkaline soil of Eastern Uttar Pradesh, India. The isolate B. cereus SIU1 was grown in modified glucose yeast extract (modified GYE) medium at pH 9.0 and 45°C. It produced maximum protease at 20 h incubation. The enzyme was ...

  12. Oxidant and solvent stable alkaline protease from Aspergillus flavus ...

    African Journals Online (AJOL)

    The increase in agricultural practices has necessitated the judicious use of agricultural wastes into value added products. In this study, an extracellular, organic solvent and oxidant stable, serine protease was produced by Aspergillus flavus MTCC 9952 under solid state fermentation. Maximum protease yield was obtained ...

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

    Directory of Open Access Journals (Sweden)

    Sandeep Kaur Saggu

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

  14. 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.

  15. Response surface methodology for production, characterization and application of solvent, salt and alkali-tolerant alkaline protease from isolated fungal strain Aspergillus niger WA 2017.

    Science.gov (United States)

    Abdel Wahab, Walaa A; Ahmed, Samia A

    2018-04-17

    Isolated strain Aspergillus niger WA 2017 was selected as potential protease producer and was identified on the basis of 18S rDNA gene homology. Optimization of protease production conditions was performed using statistical methodology. The most significant factors were identified by Plackett-Burman design (PB) and were optimized by central composite design (CCD). The enzyme production was increased by 3.6-fold with statistically optimized medium when compared to the basal medium. Based on the protease activity, 25-50% ethanol fraction exhibited the highest specific activity. The partially purified enzyme showed its highest activity (4.7-fold) after 10 min incubation at pH 10.0 and 60 °C. The enzyme was stable over a wide range of pH (7-11) and salt concentration (up to 20%). Kinetic parameters Michaelis constant (K m ) and maximum velocity (V max ) were calculated at varying casein concentrations. Additionally, thermal stability of the enzyme was substantially improved by NaCl. The enzyme showed excellent stability and compatibility in presence of organic solvents and detergents retaining 115.3 and 114.5% of its activity in presence of ethanol and Tide, respectively at 40 °C for 1 h. The results revealed that the produced enzyme was able to recover silver from used X-ray film under optimized condition using statistical methodology (CCD). Copyright © 2017. Published by Elsevier B.V.

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

    African Journals Online (AJOL)

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

  17. Intracellular alkaline proteases produced by thermoacidophiles: detection of protease heterogeneity by gelatin zymography and polymerase chain reaction (PCR)

    Energy Technology Data Exchange (ETDEWEB)

    Kocab, S.; Erdem, B. [Middle East Technical University, Ankara (Turkey). Dept. of Biological Sciences

    2002-08-01

    In this study 24 thermoacidophilic archeal and bacterial strains isolated from hot-springs and hot-soils were screened for their ability to produce intracellular alkaline proteases. The protease activities of the strains, based on azocasein hydrolysis, showed a variation from 0.6 to 5.1 U. The cell extracts of three most potent producers were further examined and it was found that their proteases exhibited maximum activity at 60-70{sup o}C and showed a pH optimum over a range of pH 7.0-8.5. Gelatin zymography revealed that two of the selected archeal strains produced multiple active SDS-resistant proteases. On the other hand, PCR amplification of alkaline serine protease gene sequences of total DNA from all isolates yielded four distinct amplification fragments of 650, 450, 400 and 300 bp, which might have been derived from different serine protease genes. (author)

  18. Genomic and exoproteomic analyses of cold- and alkaline-adapted bacteria reveal an abundance of secreted subtilisin-like proteases.

    Science.gov (United States)

    Lylloff, Jeanette E; Hansen, Lea B S; Jepsen, Morten; Sanggaard, Kristian W; Vester, Jan K; Enghild, Jan J; Sørensen, Søren J; Stougaard, Peter; Glaring, Mikkel A

    2016-03-01

    Proteases active at low temperature or high pH are used in many commercial applications, including the detergent, food and feed industries, and bacteria specifically adapted to these conditions are a potential source of novel proteases. Environments combining these two extremes are very rare, but offer the promise of proteases ideally suited to work at both high pH and low temperature. In this report, bacteria from two cold and alkaline environments, the ikaite columns in Greenland and alkaline ponds in the McMurdo Dry Valley region, Antarctica, were screened for extracellular protease activity. Two isolates, Arsukibacterium ikkense from Greenland and a related strain, Arsukibacterium sp. MJ3, from Antarctica, were further characterized with respect to protease production. Genome sequencing identified a range of potential extracellular proteases including a number of putative secreted subtilisins. An extensive liquid chromatography-tandem mass spectrometry analysis of proteins secreted by A. ikkense identified six subtilisin-like proteases as abundant components of the exoproteome in addition to other peptidases potentially involved in complete degradation of extracellular protein. Screening of Arsukibacterium genome libraries in Escherichia coli identified two orthologous secreted subtilisins active at pH 10 and 20 °C, which were also present in the A. ikkense exoproteome. Recombinant production of both proteases confirmed the observed activity. © 2016 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

  19. Nonnatural amino acid incorporation into the methionine 214 position of the metzincin Pseudomonas aeruginosa alkaline protease

    Directory of Open Access Journals (Sweden)

    Honek John F

    2005-10-01

    Full Text Available Abstract Background The alkaline protease from Pseudomonas aeruginosa (AprA is a member of the metzincin superfamily of metalloendoproteases. A key feature of these proteases is a conserved methionine-containing 1,4-tight β turn at the base of the active site zinc binding region. Results To explore the invariant methionine position in this class of protease, incorporation of a nonnatural fluorinated methionine, L-difluoromethionine (DFM, into this site was accomplished. Although overproduction of the N-terminal catalytic fragment of AprA resulted in protein aggregates which could not be resolved, successful heterologous production of the entire AprA was accomplished in the presence and absence of the nonnatural amino acid. DFM incorporation was found to only slightly alter the enzyme kinetics of AprA. In addition, differential scanning calorimetry indicated no significant alteration in the thermal stability of the modified enzyme. Conclusion Although invariant in all metzincin proteases, the methionine 214 position in AprA can be successfully replaced by the nonnatural amino acid DFM resulting in little effect on protein structure and function. This study indicates that the increased size of the methyl group by the introduction of two fluorines is still sufficiently non-sterically demanding, and bodes well for the application of DFM to biophysical studies of protein structure and function in this class of protease.

  20. Nonnatural amino acid incorporation into the methionine 214 position of the metzincin Pseudomonas aeruginosa alkaline protease

    Science.gov (United States)

    Walasek, Paula; Honek, John F

    2005-01-01

    Background The alkaline protease from Pseudomonas aeruginosa (AprA) is a member of the metzincin superfamily of metalloendoproteases. A key feature of these proteases is a conserved methionine-containing 1,4-tight β turn at the base of the active site zinc binding region. Results To explore the invariant methionine position in this class of protease, incorporation of a nonnatural fluorinated methionine, L-difluoromethionine (DFM), into this site was accomplished. Although overproduction of the N-terminal catalytic fragment of AprA resulted in protein aggregates which could not be resolved, successful heterologous production of the entire AprA was accomplished in the presence and absence of the nonnatural amino acid. DFM incorporation was found to only slightly alter the enzyme kinetics of AprA. In addition, differential scanning calorimetry indicated no significant alteration in the thermal stability of the modified enzyme. Conclusion Although invariant in all metzincin proteases, the methionine 214 position in AprA can be successfully replaced by the nonnatural amino acid DFM resulting in little effect on protein structure and function. This study indicates that the increased size of the methyl group by the introduction of two fluorines is still sufficiently non-sterically demanding, and bodes well for the application of DFM to biophysical studies of protein structure and function in this class of protease. PMID:16221305

  1. Kinetics Study of Extracellular Detergent Stable Alkaline Protease from Rhizopus oryzae

    Directory of Open Access Journals (Sweden)

    Zareena Mushtaq

    2015-04-01

    Full Text Available In this study, extracellular alkaline protease was produced from Rhizopus oryzae in submerged fermentation using dairy waste (whey as a substrate. Fermentation kinetics was studied and various parameters were optimized. The strain produced maximum protease at initial medium pH of 6.0 medium depth of 26 mm, inoculum size of 2% at incubation temperature of 35ºC for 168 h of fermentation. Alkaline protease was purified to homogeneity by ammonium sulphate fractionation followed by sephadex G-100 chromatography. The molecular mass of alkaline protease was 69 kDa determined by 10% SDS-PAGE. The optimum pH and temperature of alkaline protease was 9.0 and 40ºC, respectively. Metal profile of the enzyme showed that the enzyme was non-metallic in nature. The Km , Kcat , Vmax and Kcat/Km values of purified protease were 7.0 mg/mL, 3.8 x102S-1, 54.30 µmol/min and 54.28 s-1mg -1.mL respectively, using casein as substrate. The purified alkaline protease had stability with commercial detergents.

  2. Interfacial behavior of alkaline protease at the air-water and oil-water interfaces

    Science.gov (United States)

    Zhang, Jian; Li, Yanyan; Wang, Jing; Zhang, Yue

    2018-03-01

    The interfacial behavior of alkaline protease at the air-water and n-hexane-water interfaces was investigated using interfacial tension, dilatational rheology and dynamic light scattering. Additionally, different adsorption models which are Langmuir, Frumkin, Reorientation-A and Reorientation-R were used to fitting the data of equilibrium interfacial tension for further understanding the interfacial behavior of alkaline protease. Data fitting of the equilibrium interfacial tension was achieved by IsoFit software. The results show that the molecules arrangement of the alkaline protease at the n-hexane-water interface is more tightly than at the air-water interface. The data were further analyzed to indicate that the hydrophobic chains of alkaline protease penetrate into oil phase deeper than the air phase. Also data indicate that the electrostatic interactions and hydrophobic interactions at the n-hexane-water interface are stronger than at the air-water interface within molecules of the alkaline protease. Based on comprehensive analysis of the adsorption kinetics and interfacial rheological properties, interfacial structures mechanism of alkaline protease at n-hexane-water and air-water interfaces was proposed.

  3. 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 licheniformis NH1. ... meat Sardinelle powder; WSP, whole Sardinelle powder. ... are used as cleaning additives in detergents to facilitate.

  4. 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.

  5. 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.

  6. 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.

  7. Entrappment of alkaline protease and β-galactosidase in radiation stitched together poly-N-vinylcaprolactam

    International Nuclear Information System (INIS)

    Davidenko, T.I.; Kravchenko, I.A.

    1996-01-01

    The gel formations by poly-N-vinylcaprolactam upon its γ-irradiation by the 20-25 kGy dose as a results of partial polymer stitching together is shown, which is confirmed by the CD-and thermogravimetric data. By the alkaline protease and β-galactosidase entrapment in poly-N- vinylcaprolactam stitched together by γ-irradiation, the active preparations are obtained with 90-98 % and 30-35 % activity retained for alkaline protease and β-galactosidase, respectively. The increased stability of alkaline protease at acidic pH values and higher temperature was noted, and for β-galactosidase - the possibility of repeated use of the obtained preparation for lactose hydrolysis

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

    Digital Repository Service at National Institute of Oceanography (India)

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

    ,5]. Fungi and their enzymes from the deep-sea environment have received scant attention. Proteins and peptides constitute a substantial portion of the organic nutrients present in the deep-sea sediments as well as suspended particulate matter [6... alkaline protease using a qualitative plate assay on Czapek Dox agar (CDA) supplemented with 1% skimmed milk powder (Trade name Sagar, India). Clearance zone produced around the fungal colonies in plates indicated protease positive reaction [19...

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

    Science.gov (United States)

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

    2010-01-01

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

  10. 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.

  11. Optimization of novel and greener approach for the coproduction of uricase and alkaline protease in Bacillus licheniformis by Box-Behnken model.

    Science.gov (United States)

    Pawar, Shweta V; Rathod, Virendra K

    2018-01-02

    This study explores a novel concept of coproduction of uricase and alkaline protease by Bacillus licheniformis using single substrate in single step. Seven local bacterial strains were screened for uricase production, amongst which B. licheniformis is found to produce highest uricase along with alkaline protease. Optimization of various factors influencing maximum enzyme coproduction by B. licheniformis is performed. Maximum enzyme productivity of 0.386 U/mL uricase and 0.507 U/mL alkaline protease is obtained at 8 hr of incubation period, 1% (v/v) inoculum, and at 0.2% (w/v) uric acid when the organism is cultivated at 25°C, 180 rpm, in a media containing xylose as a carbon source, urea as a nitrogen source, and initial pH of 9.5. The statistical experimental design method of Box-Behnken was further applied to obtain optimal concentration of significant parameters such as pH (9.5), uric acid concentration (0.1%), and urea concentration (0.05%). The maximum uricase and alkaline protease production by B. licheniformis using Box-Behnken design was 0.616 and 0.582 U/mL, respectively, with 1.6- and 1.13-fold increase as compared to one factor at a time optimized media. This study will be useful to develop an economic, commercially viable, and scalable process for simultaneous production of uricase and protease enzymes.

  12. Pseudomonas syringae evades host immunity by degrading flagellin monomers with alkaline protease AprA

    NARCIS (Netherlands)

    Pel, Michiel J C; van Dijken, Anja J H; Bardoel, Bart W; Seidl, Michael F; van der Ent, Sjoerd; van Strijp, Jos A G; Pieterse, Corné M J

    Bacterial flagellin molecules are strong inducers of innate immune responses in both mammals and plants. The opportunistic pathogen Pseudomonas aeruginosa secretes an alkaline protease called AprA that degrades flagellin monomers. Here, we show that AprA is widespread among a wide variety of

  13. Pseudomonas syringae evades host Immunity by degrading flagellin monomers with alkaline protease AprA

    NARCIS (Netherlands)

    Pel, M.J.C.; Van Dijken, A.J.H.; Bardoel, B.W.; Seidl, M.F; Van der Ent, S.; Van Strijp, J.A.G.

    2014-01-01

    Bacterial flagellin molecules are strong inducers of innate immune responses in both mammals and plants. The opportunistic pathogen Pseudomonas aeruginosa secretes an alkaline protease called AprA that degrades flagellin monomers. Here, we show that AprA is widespread among a wide variety of

  14. Isolation, partial purification, biochemical characterization and detergent compatibility of alkaline protease produced by Bacillus subtilis, Alcaligenes faecalis and Pseudomonas aeruginosa obtained from sea water samples

    Directory of Open Access Journals (Sweden)

    Sarika Kedar Marathe

    2018-06-01

    Full Text Available In the current study, bacteria isolated from sea water samples of Murdeshwar, Karnataka, were screened for the production of alkaline protease by culturing them onto skim milk agar media. Of the isolated bacteria, Bacillus subtilis, Pseudomonas aeruginosa and Alcaligenes faecalis showed distinct zones of hydrolysis due to enzyme production. They were each inoculated into enzyme production media under submerged fermentation conditions at 37 °C for 48 h with a constant agitation of 120 rpm. Partial purification of alkaline protease was carried out by isoelectric precipitation. Enzyme activity was determined under varying conditions of pH, incubation temperature, different substrates, carbon and nitrogen sources and salt concentrations using sigma’s universal protease activity assay. Enzyme immobilization was carried out using 2% Sodium alginate and 0.1 M ice cold CaCl2 and its activity under varying pH, temperature conditions and detergent compatibility was assayed. Efficacy of enzyme in stain removal was tested and haemolysis was observed within of 60 s which resulted in removal of the stain. Among the three organisms, enzyme from Bacillus subtilis showed highest activity in all cases indicating that it was the most ideal organism for enzyme production. Keywords: Alkaline protease, Skim milk agar, Bacillus, Alcaligenes, Pseudomonas, Isoelectric precipitation, Protease activity, Enzyme immobilization, Detergent compatibility

  15. Characterization of an alkaline protease associated with a granulosis virus of Plodia interpunctella.

    Science.gov (United States)

    Tweeten, K A; Bulla, L A; Consigli, R A

    1978-06-01

    An alkaline protease was found to be associated with the granulosis virus of the Indian meal moth. Plodia interpunctella. The protease was located within the protein matrix of the occluded virus and hydrolyzed the major constituent of this matrix, a 28,000-dalton protein (granulin), to a mixture of polypeptides ranging in molecular weight from 10,000 to 27,000. A rapid, sensitive assay for the protease was developed using radioactively labeled granulosis virus as substrate. With this assay, the proteolytic activity could be detected by measuring the release of acid-soluble peptides from the labeled virus. The protease had a pH optimum of 10.5 and a temperature optimum of 40 degrees C and was inhibited by diisopropyl phosphorofluoridate, phenylmethylsulfonyl fluoride, and L-(1-tosylamido-2-phenyl) ethyl chloromethyl ketone. Purification of the protease from matrix protein was achieved by anion-exchange and gel permeation chromatography. The molecular weight of the isolated protease, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration, was approximately 14,000.

  16. 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.

  17. Purification and molecular characterization of subtilisin-like alkaline protease BPP-A from Bacillus pumilus strain MS-1.

    Science.gov (United States)

    Miyaji, T; Otta, Y; Nakagawa, T; Watanabe, T; Niimura, Y; Tomizuka, N

    2006-03-01

    The present study was conducted by screening zein-degrading bacteria in an attempt to obtain zein-degrading protease. Soil bacteria were screened by formation of a clear zone on zein plates. Characterization of a zein-degrading bacterium indicated a taxonomic affiliation to Bacillus pumilus, and was named MS-1 strain. The strain produced two different types of extracellular proteases, BPP-A and BPP-B. In this study, we purified and characterized BPP-A because it exhibited a higher ability to hydrolyze zein than BPP-B. When casein was used as the substrate, the optimal pH for BPP-A was 11.0. In BPP-A, zein was better substrate than casein at pH 13.0, whereas casein was better one than zein at pH 11.0. The bppA gene encoded a 383-amino acid pre-pro form of BPP-A, and mature BPP-A contained 275 amino acid residues. It was concluded that BPP-A belonged to the subtilisin family. A zein-degrading bacterium assigned to B. pumilus produced two different types of extracellular proteases, BPP-A and BPP-B. BPP-A exhibited an ability to hydrolyze zein in an extreme alkaline condition. This is a first report on screening for zein-degrading micro-organisms. The subtilisin-like protease BPP-A is possible to utilize as an industrial enzyme for the production of zein hydrolysates.

  18. Screening and characterization of alkaline protease produced by a pink pigmented facultative methylotrophic (PPFM strain, MSF 46

    Directory of Open Access Journals (Sweden)

    Shanmugam Jayashree

    2014-12-01

    Full Text Available Among the various bacterial isolates, the strain MSF 46 isolated from thorn forest soil samples, Tamil Nadu, India, was screened and characterized for its proteolytic activity. While the 16S rRNA sequencing and biochemical characterization revealed that the strain closely resembles Methylobacterium sp., methylotrophy of the strain was confirmed by the sequence homology of mxaF gene with other relative Methylobacterium sp. The alkaline protease was purified to homogeneity using DEAE cellulose ion exchange chromatography, with a 5.2-fold increase in specific activity and 34% recovery. The apparent molecular weight of the enzyme was determined as 40 kDa by SDS–PAGE study. The pH and temperature optima were 9.0 and 50 °C respectively with maximum protease activity of 1164 U/ml. Protease of MSF 46 was active in a broad pH range 7.0–11.0 with a maximum at pH 8.5 and exhibited thermostability at 50 °C. The enzyme activity was inhibited by PMSF but showed stability with Tween 20, Triton X-100 and hydrogen peroxide. Nearly 30% reduction in enzyme activity was observed in the presence of EDTA and DTT. The enzyme was effective in hydrolyzing gelatin, skimmed milk and blood clots and exhibited the potency for dehairing of goat skin and removing blood stain from cotton fabric. Significant morphological changes were observed under scanning electron microscope between cells grown in normal and casein amended medium. This first detailed report on the production of alkaline protease by a PPFM strain appears promising toward development of protocols for mass production, study of the molecular mechanism and other applications.

  19. Gelatin hydrolysates from farmed Giant catfish skin using alkaline proteases and its antioxidative function of simulated gastro-intestinal digestion.

    Science.gov (United States)

    Ketnawa, Sunantha; Martínez-Alvarez, Oscar; Benjakul, Soottawat; Rawdkuen, Saroat

    2016-02-01

    This work aims to evaluate the ability of different alkaline proteases to prepare active gelatin hydrolysates. Fish skin gelatin was hydrolysed by visceral alkaline-proteases from Giant catfish, commercial trypsin, and Izyme AL®. All antioxidant activity indices of the hydrolysates increased with increasing degree of hydrolysis (Pfish skin, could serve as a potential source of functional food ingredients for health promotion. Copyright © 2015 Elsevier Ltd. All rights reserved.

  20. 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

  1. Characterisation of a detergent-stable alkaline protease from a novel thermophilic strain Paenibacillus tezpurensis sp. nov. AS-S24-II.

    Science.gov (United States)

    Rai, Sudhir K; Roy, Jetendra K; Mukherjee, Ashis K

    2010-02-01

    An alkaline-protease-producing bacterial strain (AS-S24-II) isolated from a soil sample in Assam is a Gram-stain-positive, catalase-positive, endospore-forming rod and grows at temperatures ranging from 30 degrees C to 60 degrees C and salinity ranging from 0% to 7% (w/v) NaCl. Phenotypic characterisation, chemotaxonomic properties, presence of Paenibacillus-specific signature sequences, and ribotyping data suggested that the strain AS-S24-II represents a novel species of the genus Paenibacillus, for which the name Paenibacillus tezpurensis sp. nov. (MTCC 8959) is proposed. Phylogenetic analysis revealed that P. lentimorbus strain DNG-14 and P. lentimorbus strain DNG-16 represent the closest phylogenetic neighbour of this novel strain. Alkaline protease production (598 x 10(3) U l(-1)) by P. tezpurensis sp. nov. in SmF was optimised by response surface method. A laundry-detergent-stable, Ca(2+)-independent, 43-kDa molecular weight alkaline serine protease from this strain was purified with a 1.7-fold increase in specific activity. The purified protease displayed optimum activity at pH 9.5 and 45-50 degrees C temperature range and exhibited a significant stability and compatibility with surfactants and most of the tested commercial laundry detergents at room temperature. Further, the protease improved the wash performance of detergents, thus demonstrating its feasibility for inclusion in laundry detergent formulations.

  2. 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.

  3. 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.

  4. Pseudomonas syringae evades host Immunity by degrading flagellin monomers with alkaline protease AprA

    OpenAIRE

    Pel, M.J.C.; Van Dijken, A.J.H.; Bardoel, B.W.; Seidl, M.F; Van der Ent, S.; Van Strijp, J.A.G.

    2014-01-01

    Bacterial flagellin molecules are strong inducers of innate immune responses in both mammals and plants. The opportunistic pathogen Pseudomonas aeruginosa secretes an alkaline protease called AprA that degrades flagellin monomers. Here, we show that AprA is widespread among a wide variety of bacterial species. In addition, we investigated the role of AprA in virulence of the bacterial plant athogen P. syringae pv. tomato DC3000. The AprA-deficient DC3000 ΔaprA knockout mutant was significantl...

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

    African Journals Online (AJOL)

    The present study is concerned with the selection of new economical media based on agricultural and marine-processing by-products for the production of alkaline proteases by Bacillus licheniformis NH1. Powders from different fish species were prepared and then tested as growth media at a concentration of 10 g/l for ...

  6. A parametric study ot protease production in batch and fed-batch cultures of Bacillus firmus.

    Science.gov (United States)

    Moon, S H; Parulekar, S J

    1991-03-05

    Proteolytic enzymes produced by Bacillus species find a wide variety of applications in brewing, detergent, food, and leather industries. Owing to significant differences normally observed in culture conditions promoting cell growth and those promoting production of metabolites such as enzymes, for increased efficacy of bioreactor operations it is essential to identify these sets of conditions (including medium formulation). This study is focused on formulation of a semidefined medium that substantially enhances synthesis and secretion of an alkaline protease in batch cultures of Bacillus firmus NRS 783, a known superior producer of this enzyme. The series of experiments conducted to identify culture conditions that lead to improved protease production also enables investigation of the regulatory effects of important culture parameters including pH, dissolved oxygen, and concentrations of nitrogen and phosphorous sources and yeast extract in the medium on cell growth, synthesis and secretion of protease, and production of two major nonbiomass products, viz., acetic acid and ethanol. Cell growth and formation of the three nonbiomass products are hampered significantly under nitrogen, phosphorous, or oxygen limitation, with the cells being unable to grow in an oxygen-free environment. Improvement in protease production is achieved with respect to each culture parameter, leading in the process to 80% enhancement in protease activity over that attained using media reported in the literature. Results of a few fed-batch experiments with constant feed rate, conducted to examine possible enhancement in protease production and to further investigate repression of protease synthesis by excess of the principal carbon and nitrogen sources, are also discussed. The detailed investigation of stimulatory and repressory effects of simple and complex nutrients on protease production and metabolism of Bacillus firmus conducted in this study will provide useful guidelines for design

  7. Cloned Bacillus subtilis alkaline protease (aprA) gene showing high level of keratinolytic activity.

    Science.gov (United States)

    Zaghloul, T I

    1998-01-01

    The Bacillus subtilis alkaline protease(aprA) gene was previously cloned on a pUBHO-derivative plasmid. High levels of expression and gene stability were demonstrated when B. subtilis cells were grown on the laboratory medium 2XSG. B. subtilis cells harboring the multicopy aprA gene were grown on basal medium, supplemented with 1 % chicken feather as a source of energy, carbon, and nitrogen. Proteolytic and keratinolytic activities were monitored throughout the cultivation time. A high level of keratinolytic activity was obtained, and this indicates that alkaline protease is acting as a keratinase. Furthermore, considerable amounts of soluble proteins and free amino acids were obtained as a result of the enzymatic hydrolysis of feather. Biodegradation of feather waste using these cells represents an alternative way to improve the nutritional value of feather, since feather waste is currently utilized on a limited basis as a dietary protein supplement for animal feedstuffs. Moreover, the release of free amino acids from feather and the secreted keratinase enzyme would promote industries based on feather waste.

  8. 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...

  9. The LasB Elastase of Pseudomonas aeruginosa Acts in Concert with Alkaline Protease AprA To Prevent Flagellin-Mediated Immune Recognition.

    Science.gov (United States)

    Casilag, Fiordiligie; Lorenz, Anne; Krueger, Jonas; Klawonn, Frank; Weiss, Siegfried; Häussler, Susanne

    2016-01-01

    The opportunistic pathogen Pseudomonas aeruginosa is capable of establishing severe and persistent infections in various eukaryotic hosts. It encodes a wide array of virulence factors and employs several strategies to evade immune detection. In the present study, we screened the Harvard Medical School transposon mutant library of P. aeruginosa PA14 for bacterial factors that modulate interleukin-8 responses in A549 human airway epithelial cells. We found that in addition to the previously identified alkaline protease AprA, the elastase LasB is capable of degrading exogenous flagellin under calcium-replete conditions and prevents flagellin-mediated immune recognition. Our results indicate that the production of two proteases with anti-flagellin activity provides a failsafe mechanism for P. aeruginosa to ensure the maintenance of protease-dependent immune-modulating functions. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  10. 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.

  11. Pseudomonas syringae evades host immunity by degrading flagellin monomers with alkaline protease AprA.

    Science.gov (United States)

    Pel, Michiel J C; van Dijken, Anja J H; Bardoel, Bart W; Seidl, Michael F; van der Ent, Sjoerd; van Strijp, Jos A G; Pieterse, Corné M J

    2014-07-01

    Bacterial flagellin molecules are strong inducers of innate immune responses in both mammals and plants. The opportunistic pathogen Pseudomonas aeruginosa secretes an alkaline protease called AprA that degrades flagellin monomers. Here, we show that AprA is widespread among a wide variety of bacterial species. In addition, we investigated the role of AprA in virulence of the bacterial plant pathogen P. syringae pv. tomato DC3000. The AprA-deficient DC3000 ΔaprA knockout mutant was significantly less virulent on both tomato and Arabidopsis thaliana. Moreover, infiltration of A. thaliana Col-0 leaves with DC3000 ΔaprA evoked a significantly higher level of expression of the defense-related genes FRK1 and PR-1 than did wild-type DC3000. In the flagellin receptor mutant fls2, pathogen virulence and defense-related gene activation did not differ between DC3000 and DC3000 ΔaprA. Together, these results suggest that AprA of DC3000 is important for evasion of recognition by the FLS2 receptor, allowing wild-type DC3000 to be more virulent on its host plant than AprA-deficient DC3000 ΔaprA. To provide further evidence for the role of DC3000 AprA in host immune evasion, we overexpressed the AprA inhibitory peptide AprI of DC3000 in A. thaliana to counteract the immune evasive capacity of DC3000 AprA. Ectopic expression of aprI in A. thaliana resulted in an enhanced level of resistance against wild-type DC3000, while the already elevated level of resistance against DC3000 ΔaprA remained unchanged. Together, these results indicate that evasion of host immunity by the alkaline protease AprA is important for full virulence of strain DC3000 and likely acts by preventing flagellin monomers from being recognized by its cognate immune receptor.

  12. 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.

  13. Production of extracellular protease and glucose uptake in Bacillus clausii in steady-state and transient continuous cultures

    DEFF Research Database (Denmark)

    Christiansen, Torben; Nielsen, Jens

    2002-01-01

    The production of the extracellular alkaline protease Savinase(R) (EC 3.4.21.62) and glucose uptake in a non-sporulating strain of Bacillus clausii were investigated by analysing steady-state and transients during continuous cultivations. The specific production rate was found to have an optimum...

  14. Biomass production on saline-alkaline soils

    Energy Technology Data Exchange (ETDEWEB)

    Chaturvedi, A.N.

    1985-01-01

    In a trial of twelve tree species (both nitrogen fixing and non-fixing) for fuel plantations on saline-alkaline soil derived from Gangetic alluvium silty clay, Leucaena leucocephala failed completely after showing rapid growth for six months. Results for other species at age two showed that Prosopis juliflora had the best productivity.

  15. Improvement of acid protease production by a mixed culture of ...

    African Journals Online (AJOL)

    The synthesis of acid protease by Aspergillus oryzae AS3042 was enhanced significantly with the mixed culture of Aspergillus niger SL-09 using solid-state fermentation technique. The influence of carbon sources, nitrogen sources and the addition of phytic acid on acid protease production were investigated. The enzyme ...

  16. Pseudomonas aeruginosa alkaline protease blocks complement activation via the classical and lectin pathways.

    Science.gov (United States)

    Laarman, Alexander J; Bardoel, Bart W; Ruyken, Maartje; Fernie, Job; Milder, Fin J; van Strijp, Jos A G; Rooijakkers, Suzan H M

    2012-01-01

    The complement system rapidly detects and kills Gram-negative bacteria and supports bacterial killing by phagocytes. However, bacterial pathogens exploit several strategies to evade detection by the complement system. The alkaline protease (AprA) of Pseudomonas aeruginosa has been associated with bacterial virulence and is known to interfere with complement-mediated lysis of erythrocytes, but its exact role in bacterial complement escape is unknown. In this study, we analyzed how AprA interferes with complement activation and whether it could block complement-dependent neutrophil functions. We found that AprA potently blocked phagocytosis and killing of Pseudomonas by human neutrophils. Furthermore, AprA inhibited opsonization of bacteria with C3b and the formation of the chemotactic agent C5a. AprA specifically blocked C3b deposition via the classical and lectin pathways, whereas the alternative pathway was not affected. Serum degradation assays revealed that AprA degrades both human C1s and C2. However, repletion assays demonstrated that the mechanism of action for complement inhibition is cleavage of C2. In summary, we showed that P. aeruginosa AprA interferes with classical and lectin pathway-mediated complement activation via cleavage of C2.

  17. Water miscible mono alcohols' effect on the proteolytic performance of Bacillus clausii serine alkaline protease.

    Science.gov (United States)

    Duman, Yonca Avci; Kazan, Dilek; Denizci, Aziz Akin; Erarslan, Altan

    2014-01-01

    In this study, our investigations showed that the increasing concentrations of all examined mono alcohols caused a decrease in the Vm, kcat and kcat/Km values of Bacillus clausii GMBE 42 serine alkaline protease for casein hydrolysis. However, the Km value of the enzyme remained almost the same, which was an indicator of non-competitive inhibition. Whereas inhibition by methanol was partial non-competitive, inhibition by the rest of the alcohols tested was simple non-competitive. The inhibition constants (KI) were in the range of 1.32-3.10 M, and the order of the inhibitory effect was 1-propanol>2-propanol>methanol>ethanol. The ΔG(≠) and ΔG(≠)E-T values of the enzyme increased at increasing concentrations of all alcohols examined, but the ΔG(≠)ES value of the enzyme remained almost the same. The constant Km and ΔG(≠)ES values in the presence and absence of mono alcohols indicated the existence of different binding sites for mono alcohols and casein on enzyme the molecule. The kcat of the enzyme decreased linearly by increasing log P and decreasing dielectric constant (D) values, but the ΔG(≠) and ΔG(≠)E-T values of the enzyme increased by increasing log P and decreasing D values of the reaction medium containing mono alcohols.

  18. Screening and characterization of alkaline protease produced by a pink pigmented facultative methylotrophic (PPFM) strain, MSF 46

    OpenAIRE

    Jayashree, Shanmugam; Annapurna, Balumuri; Jayakumar, Renganathan; Sa, Tongmin; Seshadri, Sundaram

    2014-01-01

    Among the various bacterial isolates, the strain MSF 46 isolated from thorn forest soil samples, Tamil Nadu, India, was screened and characterized for its proteolytic activity. While the 16S rRNA sequencing and biochemical characterization revealed that the strain closely resembles Methylobacterium sp., methylotrophy of the strain was confirmed by the sequence homology of mxaF gene with other relative Methylobacterium sp. The alkaline protease was purified to homogeneity using DEAE cellulose ...

  19. 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.

  20. Protease Production by Different Thermophilic Fungi

    Science.gov (United States)

    Macchione, Mariana M.; Merheb, Carolina W.; Gomes, Eleni; da Silva, Roberto

    A comparative study was carried out to evaluate protease production in solid-state fermentation (SSF) and submerged fermentation (SmF) by nine different thermophilic fungi — Thermoascus aurantiacus Miehe, Thermomyces lanuginosus, T. lanuginosus TO.03, Aspergillus flavus 1.2, Aspergillus sp. 13.33, Aspergillus sp. 13.34, Aspergillus sp. 13.35, Rhizomucor pusillus 13.36 and Rhizomucor sp. 13.37 — using substrates containing proteins to induce enzyme secretion. Soybean extract (soybean milk), soybean flour, milk powder, rice, and wheat bran were tested. The most satisfactory results were obtained when using wheat bran in SSF. The fungi that stood out in SSF were T. lanuginosus, T. lanuginosus TO.03, Aspergillus sp. 13.34, Aspergillus sp. 13.35, and Rhizomucor sp. 13.37, and those in SmF were T. aurantiacus, T. lanuginosus TO.03, and 13.37. In both fermentation systems, A. flavus 1.2 and R. pusillus 13.36 presented the lowest levels of proteolytic activity.

  1. Cloning and sequencing of an alkaline protease gene from Bacillus lentus and amplification of the gene on the B. lentus chromosome by an improved technique.

    Science.gov (United States)

    Jørgensen, P L; Tangney, M; Pedersen, P E; Hastrup, S; Diderichsen, B; Jørgensen, S T

    2000-02-01

    A gene encoding an alkaline protease was cloned from an alkalophilic bacillus, and its nucleotide sequence was determined. The cloned gene was used to increase the copy number of the protease gene on the chromosome by an improved gene amplification technique.

  2. 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.

  3. Heavy water production by alkaline water electrolysis

    International Nuclear Information System (INIS)

    Kamath, Sachin; Sandeep, K.C.; Bhanja, Kalyan; Mohan, Sadhana; Sugilal, G.

    2014-01-01

    Several heavy water isotope production processes are reported in literature. Water electrolysis in combination with catalytic exchange CECE process is considered as a futuristic process to increase the throughput and reduce the cryogenic distillation load but the application is limited due to the high cost of electricity. Any improvement in the efficiency of electrolyzers would make this process more attractive. The efficiency of alkaline water electrolysis is governed by various phenomena such as activation polarization, ohmic polarization and concentration polarization in the cell. A systematic study on the effect of these factors can lead to methods for improving the efficiency of the electrolyzer. A bipolar and compact type arrangement of the alkaline water electrolyzer leads to increased efficiency and reduced inventory in comparison to uni-polar tank type electrolyzers. The bipolar type arrangement is formed when a number of single cells are stacked together. Although a few experimental studies have been reported in the open literature, CFD simulation of a bipolar compact alkaline water electrolyzer with porous electrodes is not readily available.The principal aim of this study is to simulate the characteristics of a single cell compact electrolyzer unit. The simulation can be used to predict the Voltage-Current Density (V-I) characteristics, which is a measure of the efficiency of the process.The model equations were solved using COMSOL multi-physics software. The simulated V-I characteristic is compared with the experimental data

  4. Optimizing PHB and Protease Production by Box Behnken Design

    Directory of Open Access Journals (Sweden)

    Amro Abd al fattah Amara

    2013-04-01

    Full Text Available Mixed culture is more suitable to adapt more flexible fermentation process and produce different product simultaneously. In this study a mixed Bacillus culture was investigated for their ability to produce the bioplastic "Polyhydroxybutyrate" and both of the mesophilic and the thermophilic proteases in one flask. Box-Behnken experimental design was used. The produced amount of PHB has been increased significantly. Meanwhile there is a competition between PHB and proteases. The maximum produced amount of PHB using Box-Behnken design was 2.82 g/l/48 h with protease activity equal to 41.9 Units/ml/48 h for thermophilic proteases and 99.65 Units/ml/48 h for mesophilic proteases. Excel solver was used for extra-optimization for the optimum conditions obtained from Box-Behnken experiments and its model. The maximum PHB obtained after using Excel solver was 2.88 g/l/48 h. The maximum mesophilic and thermophilic activities obtained at the same PHB production conditions were 175.68 and 243.38 Units/ml respectively. The model accuracy as obtained from Excel solver was 118.8%, which prove the power of the experimental design in optimizing such complicated process. The strategies used in this study are recommended for the production of PHB and different proteases simultaneously using Bacillus mixed culture. ABSTRAK: Kultur campuran adalah lebih sesuai bagi proses penapaian yang fleksibel dan ia boleh menghasilkan produk yang berbeza secara serentak. Dalam kajian ini keupayaan  menghasilkan "Polyhydroxybutyrate" bioplastik serta mesofilik dan termofilik protease dalam satu flask oleh  kultur Bacillus campuran telah disiasat. Eksperimen rekabentuk Box-Behnken telah digunakan. Jumlah PHB yang dikeluarkan meningkat dengan ketara dan terdapat persaingan antara PHB dan protease. Jumlah keluaran PHB maksima menggunakan rekabentuk Box-Behnken adalah 2.82 g/l/48 jam dengan aktiviti protease sama dengan 41.9 Unit/ml/48 jam untuk protease termofilik dan 99.65 Unit

  5. Detergent-compatible proteases: microbial production, properties, and stain removal analysis.

    Science.gov (United States)

    Niyonzima, Francois Niyongabo; More, Sunil

    2015-01-01

    Proteases are one of the most important commercial enzymes used in various industrial domains such as detergent and leather industries. The alkaline proteases as well as other detergent-compatible enzymes such as lipases and amylases serve now as the key components in detergent formulations. They break down various stains during fabric washing. The search for detergent-compatible proteases with better properties is a continuous exercise. The current trend is to use detergent-compatible proteases that are stable over a wide temperature range. Although the proteases showing stability at elevated pH have the capacity to be used in detergent formulations, their usage can be significant if they are also stable and compatible with detergent and detergent ingredients, and also able to remove protein stains. Despite the existence of some reviews on alkaline proteases, there is no specification for the use of alkaline proteases as detergent additives. The present review describes the detergent-compatible proteases tested as detergent additives. An overview was provided for screening, optimization, purification, and properties of detergent compatible proteases, with an emphasis on the stability and compatibility of the alkaline proteases with the detergent and detergent compounds, as well as stain removal examination methods.

  6. Deletion of aprA and nprA genes for alkaline protease A and neutral protease A from bacillus thuringiensis: effect on insecticidal crystal proteins.

    Science.gov (United States)

    Tan, Y; Donovan, W P

    2001-11-17

    The aprA gene encoding alkaline protease A (AprA) was cloned from Bacillus thuringiensis subsp. kurstaki, and the cloned gene was used to construct aprA-deleted (aprA1) strains of B. thuringiensis. An aprA1 strain of B. thuringiensis that contained the wild-type gene for neutral protease A (nprA(+)) displayed levels of extracellular proteolytic activity that were similar to those of an aprA(+)nprA(+) strain. However, when EDTA was included in the protease assay to inhibit NprA activity the aprA1nprA(+) strain displayed only 2% of the extracellular proteolytic activity of the aprA(+)nprA(+) strain. A strain that was deleted for both aprA and nprA (aprA1nprA3 strain) failed to produce detectable levels of proteolytic activity either in the presence or absence of EDTA in the assay. Compared with the aprA(+)nprA(+) strain the aprA1nprA(+) strain yielded 10% more full-length Cry1Bb crystal protein and the aprA1nprA3 strain yielded 25% more full-length Cry1Bb protein. No significant differences were seen in the 50% lethal dose of Cry1Bb protein from aprA(+)nprA(+) and aprA1nprA3 strains against three species of lepidopteran insects. These results suggest that enhanced yield of certain crystal proteins can be obtained by deletion of the genes aprA and nprA which are the major extracellular proteases of B. thuringiensis.

  7. 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.

  8. Earthworm Protease

    Directory of Open Access Journals (Sweden)

    Rong Pan

    2010-01-01

    Full Text Available The alimentary tract of earthworm secretes a group of proteases with a relative wide substrate specificity. In 1983, six isozymes were isolated from earthworm with fibrinolytic activities and called fibriniolytic enzymes. So far, more isozymes have been found from different earthworm species such as Lumbricus rubellus and Eisenia fetida. For convenience, the proteases are named on the basis of the earthworm species and the protein function, for instance, Eisenia fetida protease (EfP. The proteases have the abilities not only to hydrolyze fibrin and other protein, but also activate proenzymes such as plasminogen and prothrombin. In the light of recent studies, eight of the EfPs contain oligosaccharides chains which are thought to support the enzyme structure. Interestingly, EfP-II has a broader substrate specificity presenting alkaline trypsin, chymotrypsin and elastase activities, but EfP-III-1 has a stricter specificity. The protein crystal structures show the characteristics in their specificities. Earthworm proteases have been applied in several areas such as clinical treatment of clotting diseases, anti-tumor study, environmental protection and nutritional production. The current clinical utilizations and some potential new applications of the earthworm protease will be discussed in this paper.

  9. Earthworm Protease

    International Nuclear Information System (INIS)

    Pan, R.; Zhang, Z.; He, R.

    2010-01-01

    The alimentary tract of earthworm secretes a group of proteases with a relative wide substrate specificity. In 1983, six isozymes were isolated from earthworm with fibrinolytic activities and called fibrinolytic enzymes. So far, more isozymes have been found from different earthworm species such as Lumbricus rubellus and Eisenia fetida. For convenience, the proteases are named on the basis of the earthworm species and the protein function, for instance, Eisenia fetida protease (EfP). The proteases have the abilities not only to hydrolyze fibrin and other protein, but also activate pro enzymes such as plasminogen and prothrombin. In the light of recent studies, eight of the EfPs contain oligosaccharides chains which are thought to support the enzyme structure. Interestingly, EfP-II has a broader substrate specificity presenting alkaline trypsin, chymotrypsin and elastase activities, but EfP-III-1 has a stricter specificity. The protein crystal structures show the characteristics in their specificities. Earthworm proteases have been applied in several areas such as clinical treatment of clotting diseases, anti-tumor study, environmental protection and nutritional production. The current clinical utilizations and some potential new applications of the earthworm protease will be discussed in this paper.

  10. 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.

  11. Hydrogen production by alkaline water electrolysis

    Directory of Open Access Journals (Sweden)

    Diogo M. F. Santos

    2013-01-01

    Full Text Available Water electrolysis is one of the simplest methods used for hydrogen production. It has the advantage of being able to produce hydrogen using only renewable energy. To expand the use of water electrolysis, it is mandatory to reduce energy consumption, cost, and maintenance of current electrolyzers, and, on the other hand, to increase their efficiency, durability, and safety. In this study, modern technologies for hydrogen production by water electrolysis have been investigated. In this article, the electrochemical fundamentals of alkaline water electrolysis are explained and the main process constraints (e.g., electrical, reaction, and transport are analyzed. The historical background of water electrolysis is described, different technologies are compared, and main research needs for the development of water electrolysis technologies are discussed.

  12. Production of Microbial Protease from Selected Soil Fungal Isolates ...

    African Journals Online (AJOL)

    Production of Microbial Protease from Selected Soil Fungal Isolates. ... Nigerian Journal of Biotechnology ... and 500C. The optimal pH on the enzyme production was observed to be between pH 3.5 and 5.5 for the organisms. Keywords: Soil microorganism, fungal isolate, incubation period, microbial enzyme. Nig J. Biotech.

  13. Production of Thermostab Streptomyc ction of Thermostable Alkaline ...

    African Journals Online (AJOL)

    and temperature of 500C. Therefore ... Actinomycetes species and determining the cultural conditions se yields. ... ials for protease production on skimmed milk agar, only Norcar ... produced crude enzyme demonstrated optimal activity at pH 8.

  14. 21 CFR 184.1027 - Mixed carbohydrase and protease enzyme product.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Mixed carbohydrase and protease enzyme product. 184... RECOGNIZED AS SAFE Listing of Specific Substances Affirmed as GRAS § 184.1027 Mixed carbohydrase and protease enzyme product. (a) Mixed carbohydrase and protease enzyme product is an enzyme preparation that includes...

  15. 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.

  16. Production of extracellular aspartic protease in submerged ...

    African Journals Online (AJOL)

    Fungal milk-clotting enzymes have gained value as bovine Chymosin substitutes in the cheese industry. In this work, the effects of culture conditions on the production of extracellular milk clotting enzymes from Mucor mucedo DSM 809 in submerged fermentation were studied. The maximum activity was observed after 48 h ...

  17. Isolasi, Seleksi Dan Opttmasi Produksi Protease Daribeberapaisolat Bakteri*(isolation, Selection and Optimalization of Protease Production of Some Bacterial Isolates)

    OpenAIRE

    Naiola, Elidar; Widhyastuti, Nunuk

    2002-01-01

    Thirty-seven out of sixty-one bacterial isolates from various sources of samples were screened for protease production. The isolate of ISO PL3 could produce the highest enzyme activity, and it was used as a standard bacterial strain in this observation. For any reason,we implemented ISO PL2 to study the optimum condition for producing bacterial protease. Result shows that the maximum protease activity was obtained in a medium containing 100 gram of rice brand in a liter tofu liquid waste. The...

  18. 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.

  19. Regulation of Adrenal Aldosterone Production by Serine Protease Prostasin

    Directory of Open Access Journals (Sweden)

    Takehiro Ko

    2010-01-01

    Full Text Available A serine protease prostasin has been demonstrated to have a pivotal role in the activation of the epithelial sodium channel. Systemic administration of adenovirus carrying human prostasin gene in rats resulted in an increase in plasma prostasin and aldosterone levels. However, the mechanism by which the elevation of prostasin levels in the systemic circulation stimulated the plasma aldosterone levels remains unknown. Therefore, we examined if prostasin increases the aldosterone synthesis in a human adrenocortical cell line (H295R cells. Luciferase assay using CYP11B2 promoter revealed that prostasin significantly increased the transcriptional activity of CYP11B2. Prostasin significantly increased both CYP11B2 mRNA expression and aldosterone production in a dose-dependent manner. Surprisingly, treatment with camostat mesilate, a potent prostasin inhibitor, had no effect on the aldosterone synthesis by prostasin and also a protease-dead mutant of prostasin significantly stimulated the aldosterone production. A T-type/L-type calcium channel blocker and a protein kinase C (PKC inhibitor significantly reduced the aldosterone synthesis by prostasin. Our findings suggest a stimulatory effect of prostasin on the aldosterone synthesis by adrenal gland through the nonproteolytic action and indicate a new role of prostasin in the systemic circulation.

  20. THERMOPHILIC BACILLUS LICHENIFORMIS RBS 5 ISOLATED FROM HOT TUNISIAN SPRING CO-PRODUCING ALKALINE AND THERMOSTABLE α-AMYLASE AND PROTEASE ENZYMES

    Directory of Open Access Journals (Sweden)

    Rakia Ben Salem

    2016-06-01

    Full Text Available Bacillus licheniformis RBS 5 was isolated from thermal spring in Tunisia. The isolate coproduce α-amylase and protease enzymes. The α-amylase activity showed an optimal activity at approximately 65°C and in wide pH interval ranging from 4 to 9. This enzyme was stable over the range of 45 to 70°C after 30 min of incubation and in the pH range of 8 to 10. Protease activity was optimal; at 80°C, pH 12. This enzyme was stable until 60°C over the pH range of 10 to 12. EDTA at concentration of 5 mM reduces slightly both activities evoking the serine alkaline protease. Cationic ions (Ca2+, Cu2+, Zn2+, and Mg 2+ have an inhibition effect on α-amylase. However, protease activity was enhanced by Ca2+, Cu2+ and Mg 2+; the other cations reduce slightly the proteolytic activity. SDS and H2O2 were found as inhibitors for both activities whereas Triton X-100 and perfume have no effect. Taken together, these traits make protease activity of B. licheniformis RBS 5 as efficient for use in detergent industry.

  1. Recovery of silver from used X-ray film using alkaline protease from ...

    African Journals Online (AJOL)

    Jane Erike-Etchie

    2016-06-29

    Jun 29, 2016 ... Silver is an important industrial metal used in several areas such as photographic and x-ray films, jewelries, silver ... field of enzyme production. It is widely used in leather industry, diagnosis process, extraction of silver, animal.

  2. Photovoltaic hydrogen production with commercial alkaline electrolysers

    Energy Technology Data Exchange (ETDEWEB)

    Ursua, A.; Lopez, J.; Gubia, E.; Marroyo, L.; Sanchis, P. [Public Univ. of Navarra, Pamplona (Spain). Dept. of Electric and Electronic Engineering

    2010-07-01

    Renewable energy sources and Electrolysis generate the so-called green Hydrogen, a zero-emission and potentially fossil fuel independent energy source. However, the inherent variability of the renewable energy sources implies a mode of operation for which most current electrolysers have not been designed. This paper analyses the operation of a water electrolyser fed with photovoltaic (PV) generator electric profile. The system, Integrated by a 1 Nm{sup 3}/h Hydrogenics alkaline electrolyser and a 5100 W PV generator with 60 BP585 modules, is installed at the Public University of Navarra (Spain). The PV generator profile fed to the electrolyser is emulated by a custom-made apparatus designed and built by the authors of this paper. The profile is designed according to real irradiance data measured by a calibration cell. The irradiance data are converted to the electric power profile that the PV generator would have delivered in case of having been connected to the electrolyser by means of a DC/DC converter with maximum power point tracking (MPPT). Finally, from previously measured power-current electrolyser characteristic curves, the current profile to be delivered to the electrolyser is obtained and programmed to the electronic device. The electrolyser was tested for two types of days. During the first day, the irradiance was very stable, whereas during the second day, the irradiance was very variable. The experimental results show an average power consumption rate and an efficiency of 4908 Wh/Nm{sup 3} and 72.1%, on the first day, and 4842 Wh/Nm{sup 3} and 73.3% on the second day. The electrolyser performance was particularly good in spite of the high variability of the electric supply of the second day. (orig.)

  3. Purification and characterization of a protease from Thermophilic ...

    African Journals Online (AJOL)

    AJB SERVER

    2006-10-19

    PAGE ... applications has been well recognized and it was ... One particular interest is the production of alkaline protease from bacillus for applications in detergent industry. (Ferrero et al., 1996; Manachini and Fortina, 1998;.

  4. Cloning, characterization, expression and antifungal activity of an alkaline serine protease of Aureobasidium pullulans PL5 involved in the biological control of postharvest pathogens.

    Science.gov (United States)

    Zhang, Dianpeng; Spadaro, Davide; Valente, Silvia; Garibaldi, Angelo; Gullino, Maria Lodovica

    2012-02-15

    An alkaline protease gene was amplified from genomic DNA and cDNA of the antagonistic yeast-like fungus Aureobasidium pullulans PL5, a biocontrol agent effective against Monilinia laxa on stone fruit and Botrytis cinerea and Penicillium expansum on pome fruits. An open reading frame of 1248 bp encoding a 415-amino acid (aa) protein with a calculated molecular weight (M(r)) of 42.9 kDa and an isoelectric point (pI) of 4.5 was characterized. The cDNAALP5 gene had an 18-amino acid signal peptide, one N-gylcosylation, one histidine active site, and one serine active site. The ALP5 gene with a M(r) of 1351 bp contained two introns. One intron was of 54 bp, while the other was of 50 bp. Protein BLAST and phylogenetic tree analysis of the deduced amino sequences from the cDNAALP5 gene showed that the encoded protein had 100% homology to a protease enzyme (ALP2) of a sea strain of A. pullulans, suggesting that the protein ALP5 was an alkaline serine protease. Expression of ALP5 in Escherichia coli BL21 (DE3), followed by identification with Western-blotting, purification with Ni-NTA and analysis of enzymatic activity, yielded an homogeneous recombinant ALP5 which hydrolysed the substrate casein and inhibited the mycelial growth of the pathogens. At its optimal pH of 10.0 and reaction temperature of 50°C, the recombinant protease exhibited the highest activity towards the substrate casein, though the highest stability was at lower temperatures and pH between 7.0 and 9.0. This study provided the direct evidence that extracellular proteases secreted by the antagonist A. pullulans PL5 played a role in the biocontrol activities against some postharvest pathogens of apple and peach. Copyright © 2011 Elsevier B.V. All rights reserved.

  5. Some Investigations on Protease Enzyme Production Kinetics Using Bacillus licheniformis BBRC 100053 and Effects of Inhibitors on Protease Activity

    Directory of Open Access Journals (Sweden)

    Zahra Ghobadi Nejad

    2014-01-01

    Full Text Available Due to great commercial application of protease, it is necessary to study kinetic characterization of this enzyme in order to improve design of enzymatic reactors. In this study, mathematical modeling of protease enzyme production kinetics which is derived from Bacillus licheniformis BBRC 100053 was studied (at 37°C, pH 10 after 73 h in stationary phase, and 150 rpm. The aim of the present paper was to determine the best kinetic model and kinetic parameters for production of protease and calculating Ki (inhibition constant of different inhibitors to find the most effective one. The kinetic parameters Km (Michaelis-Menten constant and Vm (maximum rate were calculated 0.626 mM and 0.0523 mM/min. According to the experimental results, using DFP (diisopropyl fluorophosphate and PMSF (phenylmethanesulfonyl fluoride as inhibitors almost 50% of the enzyme activity could be inhibited when their concentrations were 0.525 and 0.541 mM, respectively. Ki for DFP and PMSF were 0.46 and 0.56 mM, respectively. Kinetic analysis showed that the Lineweaver-Burk model was the best fitting model for protease production kinetics DFP was more effective than PMSF and both of them should be covered in the group of noncompetitive inhibitors.

  6. Fibrinolytic protease production by new Streptomyces sp. DPUA 1576 from Amazon lichens

    Directory of Open Access Journals (Sweden)

    Germana M.M. Silva

    2015-01-01

    Conclusions: These results show that the optimization of the culture medium can enhance protease production, thus becoming a good process for further research. In addition, Streptomyces sp. DPUA 1576, isolated from Amazon lichens, might be a potential strain for fibrinolytic protease production.

  7. Hydrogen production by alkaline water electrolysis

    OpenAIRE

    Santos, Diogo M. F.; Sequeira, César A. C.; Figueiredo, José L.

    2013-01-01

    Water electrolysis is one of the simplest methods used for hydrogen production. It has the advantage of being able to produce hydrogen using only renewable energy. To expand the use of water electrolysis, it is mandatory to reduce energy consumption, cost, and maintenance of current electrolyzers, and, on the other hand, to increase their efficiency, durability, and safety. In this study, modern technologies for hydrogen production by water electrolysis have been investigated. In this article...

  8. Alkalinity production in intertidal sands intensified by lugworm bioirrigation.

    Science.gov (United States)

    Rao, Alexandra M F; Malkin, Sairah Y; Montserrat, Francesc; Meysman, Filip J R

    2014-07-05

    Porewater profiles and sediment-water fluxes of oxygen, nutrients, pH, calcium, alkalinity, and sulfide were measured in intertidal sandflat sediments from the Oosterschelde mesotidal lagoon (The Netherlands). The influence of bioturbation and bioirrigation by the deep-burrowing polychaete Arenicola marina on the rates and sources of benthic alkalinity generation was examined by comparing measurements in intact and defaunated sediment cores before and after the addition of A. marina in summer and fall 2011. Higher organic matter remineralization rates, shallower O 2 penetration, and greater sediment-water solute fluxes were observed in summer, consistent with higher sediment community metabolic rates at a higher temperature. Lugworm activity stimulated porewater exchange (5.1 × in summer, 1.9 × in fall), organic matter remineralization (6.2 × in summer, 1.9 × in fall), aerobic respiration (2.4 × in summer, 2.1 × in fall), alkalinity release (4.7 × in summer, 4.0 × in fall), nutrient regeneration, and iron cycling. The effects of lugworm activity on net sediment-water fluxes were similar but more pronounced in summer than in fall. Alkalinity release in fall was entirely driven by metabolic carbonate dissolution, while this process explained between 22 and 69% of total alkalinity production in summer, indicating the importance of other processes in this season. By enhancing organic matter remineralization and the reoxidation of reduced metabolites by the sediment microbial community, lugworm activity stimulated the production of dissolved inorganic carbon and metabolic acidity, which in turn enhanced metabolic CaCO 3 dissolution efficiency. In summer, evidence of microbial long distance electron transport (LDET) was observed in defaunated sediment. Thus, alkalinity production by net carbonate dissolution was likely supplemented by anaerobic respiration and LDET in summer.

  9. 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.

  10. Aspartic Protease Zymography Case Study: Detection of Fungal Acid Proteases by Zymography.

    Science.gov (United States)

    Kernaghan, Gavin; Mayerhofer, Michael

    2017-01-01

    This chapter describes a method for the production and characterization of fungal acid proteases. Protease production is induced by growth on BSA media over a pH gradient and protein levels are monitored over time with the Bradford assay. Once protein is depleted, the media is purified and proteases are characterized by gelatin zymography using acrylamide and buffers at near-neutral pH. Maintaining pH levels below those found in traditional zymographic systems avoids the potential loss of activity that may occur in aspartic proteases under alkaline conditions.

  11. Comparison of protease production from newly isolated bacterial ...

    African Journals Online (AJOL)

    Nasir

    2016-10-12

    Oct 12, 2016 ... Protease has gained a very important position in many industries such as food, pharmaceutical, chemical and leather industries. In this research, protease was obtained from bacteria. The bacterial strain was obtained from soil which was collected from different areas of Lahore, Pakistan. Fermentation ...

  12. Production of extracellular proteases by Mucor circinelloides using D-glucose as carbon source / substrate

    Directory of Open Access Journals (Sweden)

    Andrade Vânia Sousa

    2002-01-01

    Full Text Available Recently, some Mucorales species have been reported as protease producers. The production of extracellular proteases by Mucor circinelloides using glucose as substrate was studied. Experiments were carried out with different D-glucose concentrations (40, 60 and 80 g/L. Biomass, pH and protease activity were determined. Although biomass production had reached best yields for the medium containing D-glucose in a concentration of 80 g/L, the enzymatic production was higher when the substrate concentration was reduced to 40 g/L. The yield factor for product on cell growth and the yield factor for product on carbon substrate were higher when the microorganism grew in medium containing 40 g/L glucose. The kinetics parameters suggest that this strain seems to be promising as an alternative microorganism for protease production.

  13. Entrappment of alkaline protease and {beta}-galactosidase in radiation stitched together poly-N-vinylcaprolactam.; Vklyuchenie shchelochnoj proteazy i {beta}-galaktozidazy v radiatsionno sshityj poli-N-vinilkaprolaktam.

    Energy Technology Data Exchange (ETDEWEB)

    Davidenko, T I; Kravchenko, I A [AN Ukrainskoj SSR, Odessa (Ukraine). Fiziko-Khimicheskij Inst.

    1996-03-01

    The gel formations by poly-N-vinylcaprolactam upon its {gamma}-irradiation by the 20-25 kGy dose as a results of partial polymer stitching together is shown, which is confirmed by the CD-and thermogravimetric data. By the alkaline protease and {beta}-galactosidase entrapment in poly-N- vinylcaprolactam stitched together by {gamma}-irradiation, the active preparations are obtained with 90-98 % and 30-35 % activity retained for alkaline protease and {beta}-galactosidase, respectively. The increased stability of alkaline protease at acidic pH values and higher temperature was noted, and for {beta}-galactosidase - the possibility of repeated use of the obtained preparation for lactose hydrolysis.

  14. Dietary protease can alleviate negative effects of a coccidiosis infection on production performance in broiler chickens

    NARCIS (Netherlands)

    Peek, H.W.; Klis, van der J.D.; Vermeulen, B.; Landman, W.J.M.

    2009-01-01

    Two experiments were conducted to determine the effect of dietary protease on coccidiosis infection, production performance, the intestinal mucus layer thickness, and brush border enzyme activity using broilers challenged with Eimeria spp. laboratory isolates (Eimeria acervulina, E. maxima and E.

  15. Cloning and characterization of Bacillus subtilis iep, which has positive and negative effects on production of extracellular proteases.

    Science.gov (United States)

    Tanaka, T; Kawata, M

    1988-08-01

    We have isolated a DNA fragment from Bacillus subtilis 168 which, when present in a high-copy plasmid, inhibited production of extracellular alkaline and neutral proteases. The gene responsible for this activity was referred to as iep. The open reading frame of iep was found to be incomplete in the cloned DNA fragment. When the intact iep gene was reconstructed after the missing part of the iep gene had been cloned, it showed an enhancing effect on the production of the extracellular proteases. The open reading frame encodes a polypeptide of 229 amino acids with a molecular weight of ca. 25,866. Deletion of two amino acids from the N-terminal half of the putative iep protein resulted in dual effects, i.e., a decrease in the inhibitory activity shown by the incomplete iep gene and a slight increase in the enhancing activity shown by the complete iep gene. These results show that the iep gene product is a bifunctional protein, containing inhibitory and enhancing activities for the exoprotease production in the N-terminal and C-terminal regions, respectively. It was found by genetic and functional analyses that iep lies very close to sacU.

  16. Chemistry and biology of natural product derived protease inhibitors

    OpenAIRE

    Stolze, Sara Christina

    2012-01-01

    Im Rahmen dieser Dissertation sollten Naturstoffe und davon abgeleitete Derivate synthetisiert und im Hinblick auf ihre biologische Aktivität als Protease-Inhibitoren untersucht werden. Für die Naturstoffklasse der 3-Amino-6-Hydroxy-2-piperidon(Ahp)-Cyclodepsipeptide, die als nicht-kovalente Serin-Protease-Inhibitoren bekannt sind, konnte eine Festphasensynthese basierend auf einem allgemeinen Ahp-Vorläufermolekül entwickelt werden. Für den Ahp-Vorläufer wurde eine Lösungssynthese entwicke...

  17. Optimization of protease production by an actinomycete Strain, PS ...

    African Journals Online (AJOL)

    STORAGESEVER

    distilled water and this was inoculated into 5 ml of gelatin broth and .... leakage. After that, the dialysis bag was suspended in a beaker containing 0.5 M Tris-HCL buffer (pH 8.5) for 24 h, ... the detection of optimum temperature for the protease.

  18. A sensitive fluorescence reporter for monitoring quorum sensing regulated protease production in Vibrio harveyi.

    Science.gov (United States)

    Rajamani, Sathish; Sayre, Richard T

    2011-02-01

    Many bacteria produce and secrete proteases during host invasion and pathogenesis. Vibrio harveyi, an opportunistic pathogen of shrimp, is known to use a two-component quorum sensing (QS) mechanism for coordination of gene expression including protease secretion at high population densities. We examined the role of V. harveyi's QS signaling molecules, N-(3-hydroxybutanoyl)-L-homoserine lactone (AI-1) and the boron derivative of autoinducer-2 (BAI-2) in extracellular protease production. A fusion protein, M3CLPY (Rajamani et al., 2007), consisting of a large protease sensitive BAI-2 mutant receptor LuxP (~38kDa) flanked by two protease insensitive cyan and yellow variants of GFP (~28kDa each) was utilized as a substrate to detect secreted protease activity. The M3CLPY fusion, with the addition of wild-type V. harveyi (BB120) cell-free culture filtrate showed a time-dependent loss in fluorescence resonance energy transfer (FRET) associated with the cleavage of the LuxP linker protein and hence separation of the two fluorophores. This cleavage of LuxP linker protein leading to decreased FRET efficiency was further confirmed by immunoblotting using anti-GFP antibody. The addition of cell-free filtrates from strains defective in one or both of the two-component QS pathways: luxN(-) (defective in AI-1), luxS(-) (defective in BAI-2), and luxN(-)/luxS(-) (defective in both AI-1/BAI-2) showed differential levels of protease production. The observed protease activities were most pronounced in wild-type, followed by the AI-1 defective mutant (BB170) and the least for luxS(-) mutant (MM30) and luxN(-)/luxS(-) double mutant (MM32) strains. Incidentally, the lowest protease producing strains MM30 and MM32 were both defective in BAI-2 production. This observation was validated by addition of synthetic BAI-2 to MM30 and MM32 strains to restore protease production. Our results indicate that BAI-2 signaling in the two-component QS pathway plays the key role in regulating

  19. Digestive system development and study of acid and alkaline protease digestive capacities using biochemical and molecular approaches in totoaba (Totoaba macdonaldi) larvae.

    Science.gov (United States)

    Galaviz, Mario A; López, Lus M; García Gasca, Alejandra; Álvarez González, Carlos Alfonso; True, Conal D; Gisbert, Enric

    2015-10-01

    The present study aimed to describe and understand the development of the digestive system in totoaba (Totoaba macdonaldi) larvae from hatching to 40 days post-hatch (dph) from morphological and functional perspectives. At hatch, the digestive system of totoaba was undifferentiated. The anus and the mouth opened at 4 and 5 dph, respectively. During exogenous feeding, development of the esophagus, pancreas, liver and intestine was observed with a complete differentiation of all digestive organs. Expression and activity of trypsin and chymotrypsin were observed as early as at 1 dph, and increments in their expression and activity coincided with changes in food items (live and compound diets) and morpho-physiological development of the accessory digestive glands. In contrast, pepsin was detected later during development, which includes the appearance of the gastric glands between 24 and 28 dph. One peak in gene expression was detected at 16 dph, few days before the initial development of the stomach at 20 dph. A second peak of pepsin expression was detected at day 35, followed by a peak of activity at day 40, coinciding with the change from live to artificial food. Totoaba larvae showed a fully morphologically developed digestive system between 24 and 28 dph, as demonstrated by histological observations. However, gene expression and activity of alkaline and acid proteases were detected earlier, indicating the functionality of the exocrine pancreas and stomach before the complete morphological development of the digestive organs. These results showed that integrative studies are needed to fully understand the development of the digestive system from a morphological and functional point of views, since the histological organization of digestive structures does not reflect their real functionality. These results indicate that the digestive system of totoaba develops rapidly during the first days post-hatch, especially for alkaline proteases, and the stomach

  20. Cow dung: a potential biomass substrate for the production of detergent-stable dehairing protease by alkaliphilic Bacillus subtilis strain VV.

    Science.gov (United States)

    Vijayaraghavan, Ponnuswamy; Vijayan, Aija; Arun, Arumugaperumal; Jenisha, John Kennady; Vincent, Samuel Gnana Prakash

    2012-01-01

    Cow dung, a cheap and easily available source of energy, was used as the substrate for the production of alkaline protease by solid-state fermentation using the Bacillus subtilis strain VV. In order to achieve the maximum yield of this enzyme, the following optimum process parameters are needed: fermentation period (72 h), pH (10.0), moisture content (140%), inoculum (25%), temperature (30-40°C), carbon source (2% (w/w) maltose) and nitrogen source (1% (w/w) urea). The protease was stable over a broad temperature range (30-50°C) and pH (8.0-10.0), with maximum activity at 50°C and pH 10.0. Among the divalent ions tested, Ca(2+) (0.01 M) increased enzyme activity. The purified protease, after being subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis, was found to have a molecular mass of 38.5 kDa. The enzyme was solvent-and surfactant-stable and showed activity even after 24 h incubation along with various commercially available detergents. This enzyme possessed dehairing properties for animal hide after 16 h of incubation at room temperature. From these results it is evident that cow dung is a potential substrate for the production of a detergent-stable, dehairing protease by B. subtilis. This enzyme has a lot of potential applications in the detergent and leather-processing industries.

  1. Production, Partial Purification and Characterization of Protease From Irradiated Streptomyces Spp

    International Nuclear Information System (INIS)

    Botros, H.W.; Ahmed, A.S.

    2011-01-01

    Production and partial purification of protease by the irradiated Streptomyces spp. was the aim of this study. Streptomyces spp. was allowed to grow in culture broth of 4% shrimp shells for purpose of inducing protease enzymes. Optimal conditions for protease production were 30 degree C, 0.3 kGy, ph 7, 5x10 4 /ml inoculum size and 7 days incubation period. Protease was purified by 80% ammonium sulphate saturation which exhibited 8.7 U/ml enzyme activity. Column chromatography using sephadex G-200 exerted 23.3 U/ml enzyme activity from pooled fraction (13-16). The molecular mass of protease was determined to be 39 kDa by SDS-PAGE. The enzyme was more stable over a wide range of ph 6-8 and temperature up to 40 degree C. The produced protease was activated by Ca, Mn and FeCl 2 and completely inhibited by ethylene-diamin tetraacetic acid (EDTA) at concentration of 1000 μg/ml

  2. Draft genome sequences of two protease-producing strains of Arsukibacterium, isolated from two cold and alkaline environments

    DEFF Research Database (Denmark)

    Lylloff, Jeanette Eva; Hansen, Lea Benedicte Skov; Jepsen, Morten

    2015-01-01

    Arsukibacterium ikkense GCM72(T) and a close relative, Arsukibacterium sp. MJ3, were isolated from two cold and alkaline environments as producers of extracellular proteolytic enzymes active at high pH and low temperature. This report describes the two draft genome sequences, which may serve...

  3. 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.

  4. Production of rennin-like acid protease by Mucor pusillus through submerged fermentation

    International Nuclear Information System (INIS)

    Daudi, S.; Mukhtar, H.; Rehman, A.U.; Haq, I.U.

    2015-01-01

    The present study is concerned with the isolation and screening of Mucor species for the production of acid protease in shake flasks. Out of eight mould cultures evaluated, five were isolated from soil and three were provided from the Institute of Industrial Biotechnology, Government College University, Lahore. Of all the isolates tested, Mucor pusillus IHS6 was found to be the best producer of rennin-like acid protease producing 75 U/ml of the enzyme. Different agricultural byproducts were evaluated as fermentation substrates and maximum enzyme synthesis (61 U/ml) was obtained when rapeseed meal was used as a substrate. Optimum pH and fermentation period for the production of protease were 5.5 (56U/ml) and 72 hrs (55U/ml), respectively. The production of protease by Mucor pusillus IHS6 was also studied by adding different carbon and nitrogen sources to the fermentation medium. Fructose at a concentration of 1.5% (66 U/ml) and yeast extract at a concentration of 2% (68.2 U/ml) and ammonium chloride at a concentration of 0.1% (67U/ml) were found to be the best carbon and nitrogen (organic and inorganic) sources respectively. Spore inoculum at a concentration of 1% (68.4 U/ml) was found to be the best for protease production by Mucor pusillus. The fermentation broth was found to have strong milk clotting activity with 200 RU. (author)

  5. Production, purification and characterization of an aspartic protease from Aspergillus foetidus.

    Science.gov (United States)

    Souza, Paula Monteiro; Werneck, Gabriela; Aliakbarian, Bahar; Siqueira, Felix; Ferreira Filho, Edivaldo Ximenes; Perego, Patrizia; Converti, Attilio; Magalhães, Pérola Oliveira; Junior, Adalberto Pessoa

    2017-11-01

    An acidic thermostable protease was extracellularly produced either in shake flask or in stirred tank bioreactor by an Aspergillus foetidus strain isolated from the Brazilian savanna soil using different nitrogen sources. Its maximum activity (63.7 U mL -1 ) was obtained in a medium containing 2% (w/v) peptone. A cultivation carried out in a 5.0 L stirred-tank bioreactor provided a maximum protease activity 9% lower than that observed in Erlenmeyer flasks, which was obtained after a significantly shorter (by 16-29%) time. Protease purification by a combination of gel-filtration chromatography resulted in a 16.9-fold increase in specific activity (248.1 U g -1 ). The estimated molecular weight of the purified enzyme was 50.6 kDa, and the optimal pH and temperature were 5.0 and 55 °C, respectively. The enzyme was completely inhibited by pepstatin A, and its activity enhanced by some metals. According to the inhibition profiles, it was confirmed that the purified acid protease belongs to the aspartic protease type. These results are quite promising for future development of large-scale production of such protease, which can be useful in biotechnological applications requiring high enzyme activity and stability under acidic conditions. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. Short-chain fatty acids production and microbial community in sludge alkaline fermentation: Long-term effect of temperature.

    Science.gov (United States)

    Yuan, Yue; Liu, Ye; Li, Baikun; Wang, Bo; Wang, Shuying; Peng, Yongzhen

    2016-07-01

    Sludge alkaline fermentation has been reported to achieve efficient short-chain fatty acids (SCFAs) production. Temperature played important role in further improved SCFAs production. Long-term SCFAs production from sludge alkaline fermentation was compared between mesotherm (30±2°C) and microtherm (15±2°C). The study of 90days showed that mesotherm led to 2.2-folds production of SCFAs as microtherm and enhanced the production of acetic acid as major component of SCFAs. Soluble protein and carbohydrate at mesotherm was 2.63-folds as that at microtherm due to higher activities of protease and α-glucosidase, guaranteeing efficient substrates to produce SCFAs. Illumina MiSeq sequencing revealed that microtherm increased the abundance of Corynebacterium, Alkaliflexus, Pseudomonas and Guggenheimella, capable of enhancing hydrolysis. Hydrolytic bacteria, i.e. Alcaligenes, Anaerolinea and Ottowia, were enriched at mesotherm. Meanwhile, acidogenic bacteria showed higher abundance at mesotherm than microtherm. Therefore, enrichment of functional bacteria and higher microbial activities resulted in the improved SCFAs at mesotherm. Copyright © 2016 Elsevier Ltd. All rights reserved.

  7. Stream Insect Production as a Function of Alkalinity and Detritus Processing

    OpenAIRE

    Osborn, Thomas G.

    1981-01-01

    The study was conducted to determine if aquatic insect production was significantly different between high and low alkalinity mountain streams and if any differences were associated with food availability factors. The major objectives included determining: (1) if annual production differences occur between high and low alkalinity streams; (2) if processing rates of terrestrial detritus differs between high and low alkalinity streams; (3) if detrital processing rates are related to stream inse...

  8. Production and partial characterization of proteases from Mucor hiemalis URM3773

    Directory of Open Access Journals (Sweden)

    Roana Cecília dos Santos Ribeiro

    2015-03-01

    Full Text Available The current study evaluated the proteases production from 11 fungal species belonging to the genera Mucor, Rhizomucor and Absidia. The species were obtained from the Collection of Cultures URM at the Mycology Department-UFPE, Brazil. The best producing species was Mucor hiemalis URM 3773 (1.689 U mL-1. Plackett-Burman design methodology was employed to select the most effective parameter for protease production out of 11 medium components, including: concentration of filtrate soybean, glucose, incubation period, yeast extract, tryptone, pH, aeration, rotation, NH4Cl, MgSO4 and K2HPO4. Filtrated soybean concentration was the significant variable over the response variable, which was the specific protease activity. The crude enzyme extract showed optimal activity in pH 7.5 and at 50ºC. The enzyme was stable within a wide pH range from 5.8 to 8.0, in the phosphate buffer 0.1M and in stable temperature variation of 40-70ºC, for 180 minutes. The ions FeSO4, NaCl, MnCl2, MgCl2 and KCl stimulated the protease activity, whereas ZnCl2 ion inhibited the activity in 2.27%. Iodoacetic acid at 1mM was the proteases inhibitor that presented greater action.The results indicate that the studied enzyme have great potential for industrial application.

  9. Novel inexpensive fungi proteases: Production by solid state fermentation and characterization.

    Science.gov (United States)

    Novelli, Paula Kern; Barros, Margarida Maria; Fleuri, Luciana Francisco

    2016-05-01

    A comparative study was carried out for proteases production using agroindustrial residues as substrate for solid state fermentation (SSF) of several fungal strains. High protease production was observed for most of the microorganisms studied, as well as very different biochemical characteristics, including activities at specific temperatures and a wide range of pH values. The enzymes produced were very different regarding optimum pH and they showed stability at 50 °C. Aspergillus oryzae showed stability at all pH values studied. Penicillium roquefortii and Aspergillus flavipes presented optimum activity at temperatures of 50 °C and 90 °C, respectively. Lyophilized protease from A. oryzae reached 1251.60 U/g and yield of 155010.66 U/kg of substrate. Therefore, the substrate as well as the microorganism strain can modify the biochemical character of the enzyme produced. The high protease activity and stability established plus the low cost of substrates, make these fungal proteases potential alternatives for the biotechnological industry. Copyright © 2015 Elsevier Ltd. All rights reserved.

  10. Triclabendazole Effect on Protease Enzyme Activity in the Excretory- Secretory Products of Fasciola hepatica in Vitro.

    Directory of Open Access Journals (Sweden)

    Yosef Shrifi

    2014-03-01

    Full Text Available Fasciola hepatica is one of the most important helminthes parasites and triclabendazole (TCBZ is routinely used for treatment of infected people and animals. Secreted protease enzymes by the F. hepatica plays a critical role in the invasion, migration, nutrition and the survival of parasite and are key targets for novel drugs and vaccines. The aim of study was to determine the protease activity of excretory- secretory products (ESP of F. hepatica in the presence of TCBZ anthelmintic.F. hepatica helminthes were collected and cultured within RPMI 1640 [TCBZ treated (test and untreated (control] for 6 h at 37 °C. ESP of treated and control were collected, centrifuged and supernatants were stored at -20°C. Protein concentrations were measured according to Bradford method. Protease enzymes activities of ESP samples were estimated by using sigma's non-specific protease activity assay. ESP protein bands were detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE.Mean protein concentrations in control and treated of ESP samples were determined 196.1 ±14.52 and 376.4 ±28.20 μg/ml, respectively. Mean protease enzymes activities in control and treated were 0.37 ±0.1 and 0.089 ±0.03 U/ml, respectively. Significant difference between proteins concentrations and protease enzymes activities of two groups was observed (P<0.05. SDS-PAGE showed different patterns of protein bands between treated and control samples.The TCBZ reduced secreted protease enzymes activities and possibly effects on invasion, migration, nutrition and particularly survival of the parasite in the host tissues.

  11. Optimized production and characterization of a detergent-stable protease from Lysinibacillus fusiformis C250R.

    Science.gov (United States)

    Mechri, Sondes; Kriaa, Mouna; Ben Elhoul Berrouina, Mouna; Omrane Benmrad, Maroua; Zaraî Jaouadi, Nadia; Rekik, Hatem; Bouacem, Khelifa; Bouanane-Darenfed, Amel; Chebbi, Alif; Sayadi, Sami; Chamkha, Mohamed; Bejar, Samir; Jaouadi, Bassem

    2017-08-01

    In this study, we aimed to optimize the cultural and nutritional conditions for protease production by Lysinibacillus fusiformis strain C250R in submerged fermentation process using statistical methodology. The most significant factors (gruel, wheat bran, yeast extract, and FeSO 4 ) were identified by Plackett-Burman design. Response surface methodology (RSM) was used to determine the optimum levels of the screened factors and their interaction. Under the optimized conditions, protease yield 3100U/mL was 4.5 folds higher than those obtained by the use of the initial conditions (680U/mL). Additionally, a new extracellular 51kDa-protease, designated SAPLF, was purified and biochemically characterized from strain C250R. It shows optimum activity at 70°C and pH 10. Its half-life times at 70 and 80°C were 10 and 6-h, respectively. Irreversible inhibition of enzyme activity of SAPLF with serine protease inhibitors demonstrated that it belongs to the serine protease family. Interestingly, its catalytic efficiency was higher than that of SPVP from Aeribacillus pallidus strain VP3 and Alcalase Ultra 2.5L from Bacillus licheniformis. This study demonstrated that SAPLF has a high detergent compatibility and an excellent stain removal compared to Alcalase Ultra 2.5L; which offers an interesting potential for its application in the laundry detergent industry. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Ethanol production from bamboo using mild alkaline pre-extraction followed by alkaline hydrogen peroxide pretreatment.

    Science.gov (United States)

    Yuan, Zhaoyang; Wen, Yangbing; Kapu, Nuwan Sella

    2018-01-01

    A sequential two-stage pretreatment process comprising alkaline pre-extraction and alkaline hydrogen peroxide pretreatment (AHP) was investigated to convert bamboo carbohydrates into bioethanol. The results showed that mild alkaline pre-extraction using 8% (w/w) sodium hydroxide (NaOH) at 100°C for 180min followed by AHP pretreatment with 4% (w/w) hydrogen peroxide (H 2 O 2 ) was sufficient to generate a substrate that could be efficiently digested with low enzyme loadings. Moreover, alkali pre-extraction enabled the use of lower H 2 O 2 charges in AHP treatment. Two-stage pretreatment followed by enzymatic hydrolysis with only 9FPU/g cellulose led to the recovery of 87% of the original sugars in the raw feedstock. The use of the pentose-hexose fermenting Saccharomyces cerevisiae SR8u strain enabled the utilization of 95.7% sugars in the hydrolysate to reach 4.6%w/v ethanol titer. The overall process also enabled the recovery of 62.9% lignin and 93.8% silica at high levels of purity. Copyright © 2017 Elsevier Ltd. All rights reserved.

  13. Ecological costs and benefits correlated with trypsin protease inhibitor production in Nicotiana attenuata

    NARCIS (Netherlands)

    Glawe, G.A.; Zavala, J.A.; Kessler, A.; Van Dam, N.M.; Baldwin, I.T.

    2003-01-01

    Genotypes of the wild tobacco Nicotiana attenuata from different geographic regions in North America vary considerably in the level of constitutive and inducible trypsin protease inhibitors (TrypPIs), a potent direct defense, as well as in the production of herbivore-induced volatiles that function

  14. Effects of cysteine on growth, protease production, and catalase activity of Pseudomonas fluorescens.

    OpenAIRE

    Himelbloom, B H; Hassan, H M

    1986-01-01

    Cysteine inhibits growth of and protease production by Pseudomonas fluorescens NC3. Catalase activity in P. fluorescens NC3 was increased by cysteine. The addition of exogenous hydrogen peroxide did not increase catalase activity, thus suggesting a role for the endogenous generation of hydrogen peroxide via the autoxidation of cysteine.

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

    Science.gov (United States)

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

    2017-12-01

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

  16. Approach of describing dynamic production of volatile fatty acids from sludge alkaline fermentation.

    Science.gov (United States)

    Wang, Dongbo; Liu, Yiwen; Ngo, Huu Hao; Zhang, Chang; Yang, Qi; Peng, Lai; He, Dandan; Zeng, Guangming; Li, Xiaoming; Ni, Bing-Jie

    2017-08-01

    In this work, a mathematical model was developed to describe the dynamics of fermentation products in sludge alkaline fermentation systems for the first time. In this model, the impacts of alkaline fermentation on sludge disintegration, hydrolysis, acidogenesis, acetogenesis, and methanogenesis processes are specifically considered for describing the high-level formation of fermentation products. The model proposed successfully reproduced the experimental data obtained from five independent sludge alkaline fermentation studies. The modeling results showed that alkaline fermentation largely facilitated the disintegration, acidogenesis, and acetogenesis processes and severely inhibited methanogenesis process. With the pH increase from 7.0 to 10.0, the disintegration, acidogenesis, and acetogenesis processes respectively increased by 53%, 1030%, and 30% while methane production decreased by 3800%. However, no substantial effect on hydrolysis process was found. The model also indicated that the pathway of acetoclastic methanogenesis was more severely inhibited by alkaline condition than that of hydrogentrophic methanogenesis. Copyright © 2017 Elsevier Ltd. All rights reserved.

  17. Disruption of ten protease genes in the filamentous fungus Aspergillus oryzae highly improves production of heterologous proteins.

    Science.gov (United States)

    Yoon, Jaewoo; Maruyama, Jun-ichi; Kitamoto, Katsuhiko

    2011-02-01

    Proteolytic degradation by secreted proteases into the culture medium is one of the significant problems to be solved in heterologous protein production by filamentous fungi including Aspergillus oryzae. Double (tppA, and pepE) and quintuple (tppA, pepE, nptB, dppIV, and dppV) disruption of protease genes enhanced human lysozyme (HLY) and bovine chymosin (CHY) production by A. oryzae. In this study, we used a quintuple protease gene disruptant and performed successive rounds of disruption for five additional protease genes (alpA, pepA, AopepAa, AopepAd, and cpI), which were previously investigated by DNA microarray analyses for their expression. Gene disruption was performed by pyrG marker recycling with a highly efficient gene-targeting background (∆ligD) as previously reported. As a result, the maximum yields of recombinant CHY and HLY produced by a decuple protease gene disruptant were approximately 30% and 35%, respectively, higher than those produced by a quintuple protease gene disruptant. Thus, we successfully constructed a decuple protease gene disruptant possessing highly improved capability of heterologous protein production. This is the first report on decuple protease gene disruption that improved the levels of heterologous protein production by the filamentous fungus A. oryzae.

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

    Science.gov (United States)

    Jokela, Jouni; Heinilä, Lassi M. P.; Shishido, Tânia K.; Wahlsten, Matti; Fewer, David P.; Fiore, Marli F.; Wang, Hao; Haapaniemi, Esa; Permi, Perttu; Sivonen, Kaarina

    2017-01-01

    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. PMID:29062311

  19. 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.

  20. Study on the relationship of protease production and luminescence in Vibrio harveyi.

    Science.gov (United States)

    Nakayama, T; Nomura, N; Matsumura, M

    2006-07-01

    To demonstrate that Vibrio harveyi produces various types of toxins and how the production of those toxins is related with luminescence. Luminescence and toxicity of eight V. harveyi were evaluated. We demonstrated that all V. harveyi emitting luminescence were isolated from marine organisms and also showed that they were highly pathogenic when compared with culture collection V. harveyi based on cytotoxic assay test. On the contrary, V. harveyi isolated from shrimp farm showed no luminescence but showed high pathogenicity based on toxicity test. The effect of protease inhibitors on pathogenicity and luminescence was also investigated. We demonstrated that light emission of pathogenic V. harveyi remarkably decreased after addition of protease inhibitor. Furthermore, extracellular proteins from cell-free culture supernatant of luminescent and nonluminescent V. harveyi were compared using SDS-PAGE analysis. Results showed that there were differences in molecular weight and amount of proteins. Vibrio harveyi parasiting marine organisms have both luminescence and pathogenicity. Based on this study, luminescence and protease toxin activity in V. harveyi are related. Moreover, this paper clarified that V. harveyi produces various types of toxins. The current study demonstrated that V. harveyi produces two kinds of toxins, haemolysin and protease toxin. It may be clear roots of V. harveyi toxin.

  1. Alkalinity production in intertidal sands intensified by lugworm bioirrigation

    NARCIS (Netherlands)

    Rao, A.M.F.; Malkin, S.Y.; Montserrat, F; Meysman, F.J.R.

    2014-01-01

    Porewater profiles and sediment-water fluxes of oxygen, nutrients, pH, calcium, alkalinity, and sulfide were measured in intertidal sandflat sediments from the Oosterschelde mesotidal lagoon (The Netherlands). The influence of bioturbation and bioirrigation by the deep-burrowing polychaete

  2. Production and Characterization of an Extracellular Acid Protease from Thermophilic Brevibacillus sp. OA30 Isolated from an Algerian Hot Spring

    Directory of Open Access Journals (Sweden)

    Mohamed Amine Gomri

    2018-04-01

    Full Text Available Proteases have numerous biotechnological applications and the bioprospection for newly-thermostable proteases from the great biodiversity of thermophilic microorganisms inhabiting hot environments, such as geothermal sources, aims to discover more effective enzymes for processes at higher temperatures. We report in this paper the production and the characterization of a purified acid protease from strain OA30, a moderate thermophilic bacterium isolated from an Algerian hot spring. Phenotypic and genotypic study of strain OA30 was followed by the production of the extracellular protease in a physiologically-optimized medium. Strain OA30 showed multiple extracellular proteolytic enzymes and protease 32-F38 was purified by chromatographic methods and its biochemical characteristics were studied. Strain OA30 was affiliated with Brevibacillus thermoruber species. Protease 32-F38 had an estimated molecular weight of 64.6 kDa and was optimally active at 50 °C. It showed a great thermostability after 240 min and its optimum pH was 6.0. Protease 32-F38 was highly stable in the presence of different detergents and solvents and was inhibited by metalloprotease inhibitors. The results of this work suggest that protease 32-F38 might have interesting biotechnological applications.

  3. Production and Characterization of an Extracellular Acid Protease from Thermophilic Brevibacillus sp. OA30 Isolated from an Algerian Hot Spring.

    Science.gov (United States)

    Gomri, Mohamed Amine; Rico-Díaz, Agustín; Escuder-Rodríguez, Juan-José; El Moulouk Khaldi, Tedj; González-Siso, María-Isabel; Kharroub, Karima

    2018-04-12

    Proteases have numerous biotechnological applications and the bioprospection for newly-thermostable proteases from the great biodiversity of thermophilic microorganisms inhabiting hot environments, such as geothermal sources, aims to discover more effective enzymes for processes at higher temperatures. We report in this paper the production and the characterization of a purified acid protease from strain OA30, a moderate thermophilic bacterium isolated from an Algerian hot spring. Phenotypic and genotypic study of strain OA30 was followed by the production of the extracellular protease in a physiologically-optimized medium. Strain OA30 showed multiple extracellular proteolytic enzymes and protease 32-F38 was purified by chromatographic methods and its biochemical characteristics were studied. Strain OA30 was affiliated with Brevibacillus thermoruber species. Protease 32-F38 had an estimated molecular weight of 64.6 kDa and was optimally active at 50 °C. It showed a great thermostability after 240 min and its optimum pH was 6.0. Protease 32-F38 was highly stable in the presence of different detergents and solvents and was inhibited by metalloprotease inhibitors. The results of this work suggest that protease 32-F38 might have interesting biotechnological applications.

  4. Production, Characterization and Antioxidant Potential of Protease from Streptomyces sp. MAB18 Using Poultry Wastes

    Directory of Open Access Journals (Sweden)

    Panchanathan Manivasagan

    2013-01-01

    Full Text Available Poultry waste is an abundant renewable source for the recovery of several value-added metabolites with potential industrial applications. This study describes the production of protease on poultry waste, with the subsequent use of the same poultry waste for the extraction of antioxidants. An extracellular protease-producing strain was isolated from Cuddalore coast, India, and identified as Streptomyces sp. MAB18. Its protease was purified 17.13-fold with 21.62% yield with a specific activity of 2398.36 U/mg and the molecular weight was estimated as 43 kDa. The enzyme was optimally active at pH 8–10 and temperature 50–60°C and it was most stable up to pH 12 and 6–12% of NaCl concentration. The enzyme activity was reduced when treated with Hg2+, Pb2+, and SDS and stimulated by Fe2+, Mg2+, Triton X-100, DMSO (dimethyl sulfoxide, sodium sulphite, and β-mercaptoethanol. Furthermore, the antioxidant activities of protease were evaluated using in vitro antioxidant assays, such as DPPH radical-scavenging activity, O2 scavenging activity, NO scavenging activity, Fe2+ chelating activity, and reducing power. The enzyme showed important antioxidant potential with an IC50 value of 78±0.28 mg/mL. Results of the present study indicate that the poultry waste-derived protease may be useful as supplementary protein and antioxidant in the animal feed formulations.

  5. Optimization of Protease Production by Psychrotrophic Rheinheimera sp. with Response Surface Methodology

    OpenAIRE

    Mrayam Mahjoubin-Tehran; Bahar Shahnavaz; Razie Ghazi-Birjandi; Mansour Mashreghi; Jamshid Fooladi

    2016-01-01

    Background and Objectives: Psychrotrophic bacteria can produce enzymes at low temperatures; this provides a wide biotechnological potential, and offers numerous economical advantages over the use of mesophilic bacteria. In this study, extracellular protease production by psychrotrophic Rheinheimera sp. (KM459533) was optimized by the response surface methodology.Materials and Methods: The culture medium was tryptic soy broth containing 1% (w v -1 ) skim milk. First, the effects of variables w...

  6. Optimization of Protease Production by Psychrotrophic Rheinheimera sp. with Response Surface Methodology

    Directory of Open Access Journals (Sweden)

    Mrayam Mahjoubin-Tehran

    2016-10-01

    Full Text Available Background and Objectives: Psychrotrophic bacteria can produce enzymes at low temperatures; this provides a wide biotechnological potential, and offers numerous economical advantages over the use of mesophilic bacteria. In this study, extracellular protease production by psychrotrophic Rheinheimera sp. (KM459533 was optimized by the response surface methodology.Materials and Methods: The culture medium was tryptic soy broth containing 1% (w v -1 skim milk. First, the effects of variables were independently evaluated on the microbial growth and protease production by one-factor-at-a-time method within the following ranges: incubation time 24-120 h, temperature 15-37°C, pH 6- 11, skim milk concentration 0-2% (w v -1 , and inoculum size 0.5-3% (v v -1 . The combinational effects of the four major variable including temperature, pH, skim milk concentration, and inoculum size were then evaluated within 96 h using response surface methodology through 27 experiments.Results and Conclusion: In one-factor-at-a-time method, high cell density was detected at 72h, 20°C, pH 7, skim milk 2% (w v -1 , and inoculum size 3% (v v -1 , and maximum enzyme production (533.74 Uml-1 was achieved at 96h, 20°C, pH 9, skim milk 1% (w v -1 , and inoculum size 3% (v v -1 . The response surface methodology study showed that pH is the most effective factor in enzyme production, and among the other variables, only temperature had significant interaction with pH and inoculum size. The determination coefficient (R2 =0.9544 and non-significant lack of fit demonstrated correlation between the experimental and predicted values. The optimal conditions predicted by the response surface methodology for protease production were defined as: 22C, pH 8.5, skim milk 1.1% (w v -1 , and inoculum size 4% (v v -1 . Protease production under these conditions reached to 567.19 Uml-1 . The use of response surface methodology in this study increased protease production by eight times as

  7. Enhanced production of protease by mutagenized strain of aspergillus oryzae in solid substrate fermentation of rice bran

    International Nuclear Information System (INIS)

    Yousif, M.; Irfan, M.; Baig, S.; Iqbal, A.

    2010-01-01

    Neutral protease activity of parent strain of Aspergellus oryzae was enhanced by UV and chemical mutagenization with ethyl methane sulphonate (EMS). After screening, a hyper producing strain was isolated and found effective for tile production of neutral protease as compared to the parent strain of Aspergellus oryzae. Solid substrate fermentation was carried out in 250ml conical flask with 45 % initial moisture contents at a temperature of 30 deg. C for 72 flours. Under the optimum conditions maximum yield of neutral protease obtained was 662.61+-0.36 U/gds, Almost all the organic nitrogen supplements favored the enzyme production while sucrose proved as a best carbon source. (author)

  8. Activity of the Human Rhinovirus 3C Protease Studied in Various Buffers, Additives and Detergents Solutions for Recombinant Protein Production.

    Directory of Open Access Journals (Sweden)

    Raheem Ullah

    Full Text Available Proteases are widely used to remove affinity and solubility tags from recombinant proteins to avoid potential interference of these tags with the structure and function of the fusion partner. In recent years, great interest has been seen in use of the human rhinovirus 3C protease owing to its stringent sequence specificity and enhanced activity. Like other proteases, activity of the human rhinovirus 3C protease can be affected in part by the buffer components and additives that are generally employed for purification and stabilization of proteins, hence, necessitate their removal by tedious and time-consuming procedures before proteolysis can occur. To address this issue, we examined the effect of elution buffers used for common affinity based purifications, salt ions, stability/solubility and reducing agents, and detergents on the activity of the human rhinovirus 3C protease using three different fusion proteins at 4°C, a temperature of choice for purification of many proteins. The results show that the human rhinovirus 3C protease performs better at 4°C than the frequently used tobacco etch virus protease and its activity was insensitive to most of the experimental conditions tested. Though number of fusion proteins tested is limited, we expect that these finding will facilitate the use of the human rhinovirus 3C protease in recombinant protein production for pharmaceutical and biotechnological applications.

  9. Activity of the Human Rhinovirus 3C Protease Studied in Various Buffers, Additives and Detergents Solutions for Recombinant Protein Production.

    Science.gov (United States)

    Ullah, Raheem; Shah, Majid Ali; Tufail, Soban; Ismat, Fouzia; Imran, Muhammad; Iqbal, Mazhar; Mirza, Osman; Rhaman, Moazur

    2016-01-01

    Proteases are widely used to remove affinity and solubility tags from recombinant proteins to avoid potential interference of these tags with the structure and function of the fusion partner. In recent years, great interest has been seen in use of the human rhinovirus 3C protease owing to its stringent sequence specificity and enhanced activity. Like other proteases, activity of the human rhinovirus 3C protease can be affected in part by the buffer components and additives that are generally employed for purification and stabilization of proteins, hence, necessitate their removal by tedious and time-consuming procedures before proteolysis can occur. To address this issue, we examined the effect of elution buffers used for common affinity based purifications, salt ions, stability/solubility and reducing agents, and detergents on the activity of the human rhinovirus 3C protease using three different fusion proteins at 4°C, a temperature of choice for purification of many proteins. The results show that the human rhinovirus 3C protease performs better at 4°C than the frequently used tobacco etch virus protease and its activity was insensitive to most of the experimental conditions tested. Though number of fusion proteins tested is limited, we expect that these finding will facilitate the use of the human rhinovirus 3C protease in recombinant protein production for pharmaceutical and biotechnological applications.

  10. Coupling the Alkaline-Surfactant-Polymer Technology and The Gelation Technology to Maximize Oil Production

    Energy Technology Data Exchange (ETDEWEB)

    Malcolm Pitts; Jie Qi; Dan Wilson; Phil Dowling; David Stewart; Bill Jones

    2005-12-01

    Performance and produced polymer evaluation of four alkaline-surfactant-polymer projects concluded that only one of the projects could have benefited from combining the alkaline-surfactant-polymer and gelation technologies. Cambridge, the 1993 Daqing, Mellott Ranch, and the Wardlaw alkaline-surfacant-polymer floods were studied. An initial gel treatment followed by an alkaline-surfactant-polymer flood in the Wardlaw field would have been a benefit due to reduction of fracture flow. Numerical simulation demonstrated that reducing the permeability of a high permeability zone of a reservoir with gel improved both waterflood and alkaline-surfactant-polymer flood oil recovery. A Minnelusa reservoir with both A and B sand production was simulated. A and B sands are separated by a shale layer. A sand and B sand waterflood oil recovery was improved by 196,000 bbls or 3.3% OOIP when a gel was placed in the B sand. Alkaline-surfactant-polymer flood oil recovery improvement over a waterflood was 392,000 bbls or 6.5% OOIP. Placing a gel into the B sand prior to an alkaline-surfactant-polymer flood resulted in 989,000 bbl or 16.4% OOIP more oil than only water injection. A sand and B sand alkaline-surfactant-polymer flood oil recovery was improved by 596,000 bbls or 9.9% OOIP when a gel was placed in the B sand.

  11. Effect of alkaline microwaving pretreatment on anaerobic digestion and biogas production of swine manure

    OpenAIRE

    Tao Yu; Yihuan Deng; Hongyu Liu; Chunping Yang; Bingwen Wu; Guangming Zeng; Li Lu; Fumitake Nishimura

    2017-01-01

    Microwave assisted with alkaline (MW-A) condition was applied in the pretreatment of swine manure, and the effect of the pretreatment on anaerobic treatment and biogas production was evaluated in this study. The two main microwaving (MW) parameters, microwaving power and reaction time, were optimized for the pretreatment. Response surface methodology (RSM) was used to investigate the effect of alkaline microwaving process for manure pretreatment at various values of pH and energy input. Resul...

  12. Bioprocess optimization for production of thermoalkali-stable protease from Bacillus subtilis K-1 under solid-state fermentation.

    Science.gov (United States)

    Singh, Satbir; Bajaj, Bijender Kumar

    2016-10-02

    Cost-effective production of proteases, which are robust enough to function under harsh process conditions, is always sought after due to their wide industrial application spectra. Solid-state production of enzymes using agro-industrial wastes as substrates is an environment-friendly approach, and it has several advantages such as high productivity, cost-effectiveness, being less labor-intensive, and less effluent production, among others. In the current study, different agro-wastes were employed for thermoalkali-stable protease production from Bacillus subtilis K-1 under solid-state fermentation. Agricultural residues such as cotton seed cake supported maximum protease production (728 U ml(-1)), which was followed by gram husk (714 U ml(-1)), mustard cake (680 U ml(-1)), and soybean meal (653 U ml(-1)). Plackett-Burman design of experiment showed that peptone, moisture content, temperature, phosphates, and inoculum size were the significant variables that influenced the protease production. Furthermore, statistical optimization of three variables, namely peptone, moisture content, and incubation temperature, by response surface methodology resulted in 40% enhanced protease production as compared to that under unoptimized conditions (from initial 728 to 1020 U ml(-1)). Thus, solid-state fermentation coupled with design of experiment tools represents a cost-effective strategy for production of industrial enzymes.

  13. Effect of pH on the production of alkaline proteinase by alkalophilic Bacillus sp

    International Nuclear Information System (INIS)

    Kitada, Makio; Horikoshi, Koki

    1976-01-01

    The effect of the pH of the medium on the microbial growth and alkaline proteinase production, and on the uptake of various substances by alkalophilic Bacillus sp. No.8-1 were studied to investigate the physiological properties of alkalophilic bacteria. Both the microbial growth and alkaline proteinase production by replacement culture were maximum between pH 9 and 10. The alkaline proteinase production sources were also effective for the production. The uptake of various substances such as glucose, acetate, amino acids, and uracil, necessary for proteinase production by this strain, was maximum between pH 9 and 10. The uptake of α-aminoisobutyric acid, a nonmetabolizable amino acid analogue, was also maximum at pH 10. The pH-dependence of these substance was not due to their ionic forms being affected by extracellular pH. It was concluded from above results that good production of alkaline proteinase in alkaline media was due to the active uptake of various nutrients in this culture condition. (auth.)

  14. Enhanced biogas production from penicillin bacterial residue by thermal-alkaline pretreatment

    International Nuclear Information System (INIS)

    Zhong, Weizhang; Li, Guixia; Gao, Yan; Li, Zaixing; Geng, Xiaoling; Li, Yubing; Yang, Jingliang; Zhou, Chonghui

    2015-01-01

    In this study, the orthogonal experimental design was used to determine the optimum conditions for the effect of thermal alkaline; pretreatment on the anaerobic digestion of penicillin bacterial residue. The biodegradability of the penicillin; bacterial residue was evaluated by biochemical methane potential tests in laboratory. The optimum values of temperature,; alkali concentration, pretreatment time and moisture content for the thermal-alkaline pretreatment were determined as; 70 °C, 6% (w/v), 30 min, and 85%, respectively. Thermal-alkaline pretreatment could significantly enhance the soluble; chemical oxygen demand solubilization, the suspended solid solubilization and the biodegradability. Biogas production; was enhanced by the thermal-alkaline pretreatment, probably as a result of the breakdown of cell walls and membranes of; micro-organisms, which may facilitate the contact between organic molecules and anaerobic microorganisms.; Keywords: penicillin bacterial residue; anaerobic digestion; biochemical methane potential tests; pretreatment

  15. Optimization of Protease Production from Aspergillus Oryzae Sp. Using Box-Behnken Experimental Design

    Directory of Open Access Journals (Sweden)

    G. Srinu Babu

    2007-01-01

    Full Text Available Protease production by Aspergillus oryzae was optimized in shake-flask cultures using Box-Behnken experimental design. An empirical model was developed through response surface methodology to describe the relationship between tested variable (peptone, glucose, soyabeanmeal and pH. Maximum enzyme activity was attained with Peptone at 4 g∕L; temperature at 30 °C glucose at 6 g∕L; 30 °C and pH at 10. Experimental verification of the model showed a validation of 95%, which is more than 3-fold increase compare to the basal medium.

  16. Mesophilic and thermophilic alkaline fermentation of waste activated sludge for hydrogen production: Focusing on homoacetogenesis

    DEFF Research Database (Denmark)

    Wan, Jingjing; Jing, Yuhang; Zhang, Shicheng

    2016-01-01

    The present study compared the mesophilic and thermophilic alkaline fermentation of waste activated sludge (WAS) for hydrogen production with focus on homoacetogenesis, which mediated the consumption of H2 and CO2 for acetate production. Batch experiments showed that hydrogen yield of WAS increased...

  17. Improvement in extracellular protease production by the marine antarctic yeast Rhodotorula mucilaginosa L7.

    Science.gov (United States)

    Chaud, Luciana C S; Lario, Luciana D; Bonugli-Santos, Rafaella C; Sette, Lara D; Pessoa Junior, Adalberto; Felipe, Maria das Graças de A

    2016-12-25

    Microorganisms from extreme and restrictive eco systems, such as the Antarctic continent, are of great interest due to their ability to synthesize products of commercial value. Among these, enzymes from psychrotolerant and psychrophilic microorganisms offer potential economical benefits due to their high activity at low and moderate temperatures. The cold adapted yeast Rhodotorula mucilaginosa L7 was selected out of 97 yeasts isolated from Antarctica as having the highest extracellular proteolytic activity in preliminary tests. The present study was aimed at evaluating the effects of nutrient composition (peptone, rice bran extract, ammonium sulfate, sodium chloride) and physicochemical parameters (temperature and pH) on its proteolytic activity. A 2 6-2 fractional factorial design experiment followed by a central composite design (CCD 2 3 ) was performed to optimize the culture conditions and improve the extracellular proteolytic activity. The results indicated that the presence of peptone in the medium was the most influential factor in protease production. Enzymatic activity was enhanced by the interaction between low glucose and peptone concentrations. The optimization of culture conditions with the aid of mathematical modeling enabled a c. 45% increase in proteolytic activity and at the same time reduced the amount of glucose and peptone required for the culture. Thus culture conditions established in this work may be employed in the biotechnological production of this protease. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. Screening of Nutritional Parameters for the Production of Protease from Aspergillus Oryzae

    Directory of Open Access Journals (Sweden)

    G. Srinubabu

    2007-01-01

    Full Text Available Production of protease enzyme by fungus Aspergillus oryzae was investigated. The proteolytic activity was observed when the fungus was grown in the medium containing glucose, malt extract, yeast extract, peptone, K2HPO4, MgSO4 and FeSO4. The present paper describes the screening of media components and fermentation conditions in shake flask. The organism utilized carbon sources glucose, fructose, sucrose, lactose, dextrin and starch among them glucose was found to be the best carbon source, for nitrogen sources various inorganic and organic media components were investigated among them peptone is found to be the best nitrogen source. 1% cottonseed followed by 2% Soya bean meal was found to be the best inducer. With optimized media two-fold increase in the protease production. The fungus growth depends on the concentration of carbon, nitrogen and salt solution, where as the enzyme production was also influenced by the culture time, pH and interaction between these two variables.

  19. Immobilized protease on the magnetic nanoparticles used for the hydrolysis of rapeseed meals

    International Nuclear Information System (INIS)

    Jin Xin; Li Jufang; Huang Pingying; Dong Xuyan; Guo Lulu; Yang Liang; Cao Yuancheng; Wei Fang; Zhao Yuandi

    2010-01-01

    (3-aminopropl) triethoxysilaneand modified magnetic nanoparticles with the average diameter of 25.4 nm were synthesized in water-phase co-precipitation method. And then these nanoparticles were covalently coupled with alkaline protease as enzyme carrier by using 1,4-phenylene diisothlocyanate as coupling agent. Experiments showed that the immobilized protease can keep the catalytic bioactivity, which can reach to 47.8% when casein was served as substrate. Results showed that the catalytic activity of immobilized protease on these magnetic nanoparticles could retain 98.63±2.37% after 60 days. And it is more stable than the free protease during the shelf-life test. The enzyme reaction conditions such as optimum reaction temperature and pH are the same as free protease. Furthermore, mix-and-separate experiments showed that the immobilized protease could be recycled through the magnetic nanoparticles after the biocatalysis process. When the rapeseed meals were used as substrate, the degree of hydrolysis of immobilized alkaline protease achieved 9.86%, while it was 10.41% for the free protease. The macromolecular proteins of rapeseed meals were hydrolyzed by immobilized protease into small molecules such as polypeptides or amino acids. Thus, a novel efficient and economic way for the recycling of enzymes in the application of continuous production of active peptides was provided based on these magnetic nanoparticles.

  20. Immobilized protease on the magnetic nanoparticles used for the hydrolysis of rapeseed meals

    Energy Technology Data Exchange (ETDEWEB)

    Jin Xin [Wuhan National Laboratory for Optoelectronics-Hubei Bioinformatics and Molecular Imaging Key Laboratory, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, HuBei 430074 (China); Li Jufang [Key Lab of Oil Crops Biology, Ministry of Agriculture, Institute of Oil Crops Research, Chinese Academy of Agricultural Sciences, Wuhan, Hubei 430062 (China); Huang Pingying [Wuhan National Laboratory for Optoelectronics-Hubei Bioinformatics and Molecular Imaging Key Laboratory, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, HuBei 430074 (China); Dong Xuyan [Wuhan National Laboratory for Optoelectronics-Hubei Bioinformatics and Molecular Imaging Key Laboratory, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, HuBei 430074 (China); Key Lab of Oil Crops Biology, Ministry of Agriculture, Institute of Oil Crops Research, Chinese Academy of Agricultural Sciences, Wuhan, Hubei 430062 (China); Guo Lulu [Key Lab of Oil Crops Biology, Ministry of Agriculture, Institute of Oil Crops Research, Chinese Academy of Agricultural Sciences, Wuhan, Hubei 430062 (China); Yang Liang; Cao Yuancheng [Wuhan National Laboratory for Optoelectronics-Hubei Bioinformatics and Molecular Imaging Key Laboratory, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, HuBei 430074 (China); Wei Fang [Key Lab of Oil Crops Biology, Ministry of Agriculture, Institute of Oil Crops Research, Chinese Academy of Agricultural Sciences, Wuhan, Hubei 430062 (China); Zhao Yuandi, E-mail: zydi@mail.hust.edu.c [Wuhan National Laboratory for Optoelectronics-Hubei Bioinformatics and Molecular Imaging Key Laboratory, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, HuBei 430074 (China)

    2010-07-15

    (3-aminopropl) triethoxysilaneand modified magnetic nanoparticles with the average diameter of 25.4 nm were synthesized in water-phase co-precipitation method. And then these nanoparticles were covalently coupled with alkaline protease as enzyme carrier by using 1,4-phenylene diisothlocyanate as coupling agent. Experiments showed that the immobilized protease can keep the catalytic bioactivity, which can reach to 47.8% when casein was served as substrate. Results showed that the catalytic activity of immobilized protease on these magnetic nanoparticles could retain 98.63+-2.37% after 60 days. And it is more stable than the free protease during the shelf-life test. The enzyme reaction conditions such as optimum reaction temperature and pH are the same as free protease. Furthermore, mix-and-separate experiments showed that the immobilized protease could be recycled through the magnetic nanoparticles after the biocatalysis process. When the rapeseed meals were used as substrate, the degree of hydrolysis of immobilized alkaline protease achieved 9.86%, while it was 10.41% for the free protease. The macromolecular proteins of rapeseed meals were hydrolyzed by immobilized protease into small molecules such as polypeptides or amino acids. Thus, a novel efficient and economic way for the recycling of enzymes in the application of continuous production of active peptides was provided based on these magnetic nanoparticles.

  1. Microbial Proteases in Baked Goods: Modification of Gluten and Effects on Immunogenicity and Product Quality

    Directory of Open Access Journals (Sweden)

    Nina G. Heredia-Sandoval

    2016-08-01

    Full Text Available Gluten-related diseases are a range of inflammatory disorders of the small intestine, characterized by an adverse response to gluten ingestion; therefore, the treatment is a gluten withdrawal. In spite of the increased market of gluten-free products, widely available breads with high acceptability are still missing due to the technological challenge of substituting the special gluten properties. Instead of using alternative ingredients for baking, some attempts have been done to decrease gluten immunogenicity by its enzymatic degradation with microbial proteases. Although the gluten immunogenicity reduction has been reached to an acceptable level, some quality parameters of the products are affected. This review focus on the use of microbial peptidases to prepare less immunogenic baked goods and their effect on product quality.

  2. Microbial Proteases in Baked Goods: Modification of Gluten and Effects on Immunogenicity and Product Quality.

    Science.gov (United States)

    Heredia-Sandoval, Nina G; Valencia-Tapia, Maribel Y; Calderón de la Barca, Ana M; Islas-Rubio, Alma R

    2016-08-30

    Gluten-related diseases are a range of inflammatory disorders of the small intestine, characterized by an adverse response to gluten ingestion; therefore, the treatment is a gluten withdrawal. In spite of the increased market of gluten-free products, widely available breads with high acceptability are still missing due to the technological challenge of substituting the special gluten properties. Instead of using alternative ingredients for baking, some attempts have been done to decrease gluten immunogenicity by its enzymatic degradation with microbial proteases. Although the gluten immunogenicity reduction has been reached to an acceptable level, some quality parameters of the products are affected. This review focus on the use of microbial peptidases to prepare less immunogenic baked goods and their effect on product quality.

  3. Effect of irradiation on protease production by a Philippine strain of Aspergillus oryzae (ahlburg) cohn

    International Nuclear Information System (INIS)

    Anglo, P.G.

    1974-03-01

    The Philippine strain of Aspergillus oryzae (ahlburg) cohn. was exposed to ultraviolet rays and ionizing radiation from cobalt-60 for the purpose of obtaining possible mutants or resistant strains which produce powerful proteolytic enzymes. Out of 58 isolates, only 3 gave significant proteolytic values (PV) high enough to merit further investigation. The isolates, G-10, G-110, and 23-110, were picked from plates exposed to gamma rays from cobalt-60. Optimum incubation temperature for these isolates for highest percentage of active protease was 24 0 -27 0 C. The isolates were found capable of producing active protease from the second day of incubation up to the fifth day, whereas the activity of the parent strain was retained the fourth day only. The isolates showed maximum digestive ability at 25 0 -55 0 C, giving proteolytic values of 833. The pH activity curves showed that the enzyme produced by the irradiated isolates G-10 and G-110 were very active at pH 9.0-10.0, and isolate 23-110 at pH 6.0-10.0. The parent strain revealed two pH optima, one at pH 7.5-8.5 and the other at pH 9.0-9.5. Crude enzyme powder gave activities comparable to alkalase and maxatase, commercial proteolytic enzymes imported from Belgium and Netherlands being used as component of laundry detergents by some manufacturing companies in the Philippines. The results obtained give valuable information for the commercial application of the enzyme. Since the organism can produce high yields of protease from copra meal, a by-product of the coconut industry, commerical feasibility may be envisioned in the near future

  4. Toxicity of crude oil products and detergent on serum alkaline ...

    African Journals Online (AJOL)

    The comparative effect of exposing Clarias gariepinus juveniles (100.20 + 0.8g) to diferent concentrations of crude oil products and detergent were studied. Bonny Light Crude oil (BLCO), Premium motor spirit (PMS), Dual purpose kerosene (DPK) and Ariel Enzymax Detergent (AED) were respectively applied at three ...

  5. Approach toward enhancement of halophilic protease production by Halobacterium sp. strain LBU50301 using statistical design response surface methodology.

    Science.gov (United States)

    Chuprom, Julalak; Bovornreungroj, Preeyanuch; Ahmad, Mehraj; Kantachote, Duangporn; Dueramae, Sawitree

    2016-06-01

    A new potent halophilic protease producer, Halobacterium sp. strain LBU50301 was isolated from salt-fermented fish samples ( budu ) and identified by phenotypic analysis, and 16S rDNA gene sequencing. Thereafter, sequential statistical strategy was used to optimize halophilic protease production from Halobacterium sp. strain LBU50301 by shake-flask fermentation. The classical one-factor-at-a-time (OFAT) approach determined gelatin was the best nitrogen source. Based on Plackett - Burman (PB) experimental design; gelatin, MgSO 4 ·7H 2 O, NaCl and pH significantly influenced the halophilic protease production. Central composite design (CCD) determined the optimum level of medium components. Subsequently, an 8.78-fold increase in corresponding halophilic protease yield (156.22 U/mL) was obtained, compared with that produced in the original medium (17.80 U/mL). Validation experiments proved the adequacy and accuracy of model, and the results showed the predicted value agreed well with the experimental values. An overall 13-fold increase in halophilic protease yield was achieved using a 3 L laboratory fermenter and optimized medium (231.33 U/mL).

  6. Approach toward enhancement of halophilic protease production by Halobacterium sp. strain LBU50301 using statistical design response surface methodology

    Directory of Open Access Journals (Sweden)

    Julalak Chuprom

    2016-06-01

    Full Text Available A new potent halophilic protease producer, Halobacterium sp. strain LBU50301 was isolated from salt-fermented fish samples (budu and identified by phenotypic analysis, and 16S rDNA gene sequencing. Thereafter, sequential statistical strategy was used to optimize halophilic protease production from Halobacterium sp. strain LBU50301 by shake-flask fermentation. The classical one-factor-at-a-time (OFAT approach determined gelatin was the best nitrogen source. Based on Plackett–Burman (PB experimental design; gelatin, MgSO4·7H2O, NaCl and pH significantly influenced the halophilic protease production. Central composite design (CCD determined the optimum level of medium components. Subsequently, an 8.78-fold increase in corresponding halophilic protease yield (156.22 U/mL was obtained, compared with that produced in the original medium (17.80 U/mL. Validation experiments proved the adequacy and accuracy of model, and the results showed the predicted value agreed well with the experimental values. An overall 13-fold increase in halophilic protease yield was achieved using a 3 L laboratory fermenter and optimized medium (231.33 U/mL.

  7. Entamoeba histolytica-secreted cysteine proteases induce IL-8 production in human mast cells via a PAR2-independent mechanism.

    Science.gov (United States)

    Lee, Young Ah; Nam, Young Hee; Min, Arim; Kim, Kyeong Ah; Nozaki, Tomoyoshi; Saito-Nakano, Yumiko; Mirelman, David; Shin, Myeong Heon

    2014-01-01

    Entamoeba histolytica is an extracellular tissue parasite causing colitis and occasional liver abscess in humans. E. histolytica-derived secretory products (SPs) contain large amounts of cysteine proteases (CPs), one of the important amoebic virulence factors. Although tissue-residing mast cells play an important role in the mucosal inflammatory response to this pathogen, it is not known whether the SPs induce mast cell activation. In this study, when human mast cells (HMC-1 cells) were stimulated with SPs collected from pathogenic wild-type amoebae, interleukin IL-8 mRNA expression and production were significantly increased compared with cells incubated with medium alone. Inhibition of CP activity in the SPs with heat or the CP inhibitor E64 resulted in significant reduction of IL-8 production. Moreover, SPs obtained from inhibitors of cysteine protease (ICP)-overexpressing amoebae with low CP activity showed weaker stimulatory effects on IL-8 production than the wild-type control. Preincubation of HMC-1 cells with antibodies to human protease-activated receptor 2 (PAR2) did not affect the SP-induced IL-8 production. These results suggest that cysteine proteases in E. histolytica-derived secretory products stimulate mast cells to produce IL-8 via a PAR2-independent mechanism, which contributes to IL-8-mediated tissue inflammatory responses during the early phase of human amoebiasis. © Y.A. Lee et al., published by EDP Sciences, 2014.

  8. Entamoeba histolytica-secreted cysteine proteases induce IL-8 production in human mast cells via a PAR2-independent mechanism

    Directory of Open Access Journals (Sweden)

    Lee Young Ah

    2014-01-01

    Full Text Available Entamoeba histolytica is an extracellular tissue parasite causing colitis and occasional liver abscess in humans. E. histolytica-derived secretory products (SPs contain large amounts of cysteine proteases (CPs, one of the important amoebic virulence factors. Although tissue-residing mast cells play an important role in the mucosal inflammatory response to this pathogen, it is not known whether the SPs induce mast cell activation. In this study, when human mast cells (HMC-1 cells were stimulated with SPs collected from pathogenic wild-type amoebae, interleukin IL-8 mRNA expression and production were significantly increased compared with cells incubated with medium alone. Inhibition of CP activity in the SPs with heat or the CP inhibitor E64 resulted in significant reduction of IL-8 production. Moreover, SPs obtained from inhibitors of cysteine protease (ICP-overexpressing amoebae with low CP activity showed weaker stimulatory effects on IL-8 production than the wild-type control. Preincubation of HMC-1 cells with antibodies to human protease-activated receptor 2 (PAR2 did not affect the SP-induced IL-8 production. These results suggest that cysteine proteases in E. histolytica-derived secretory products stimulate mast cells to produce IL-8 via a PAR2-independent mechanism, which contributes to IL-8-mediated tissue inflammatory responses during the early phase of human amoebiasis.

  9. Effects of dietary protease on nitrogen emissions from broiler production: a holistic comparison using Life Cycle Assessment.

    Science.gov (United States)

    Leinonen, Ilkka; Williams, Adrian G

    2015-12-01

    The aim of the study was to quantify the effects of the use of a protease Ronozyme® ProAct in broiler feed on the environmental impacts of broiler and broiler feed production chains. This was done by using a Life Cycle Assessment (LCA) modelling approach with data from trials using both standard soya-based broiler diets and reduced-protein diets with added protease. The results for the feed production chain showed that there was a reduction in all environmental impact categories when protease was used in the diets. The biggest reduction occurred in the category of Global Warming Potential, mainly as a result of decreased carbon dioxide emissions from land use changes related to soya production. In the results for the broiler production chain, there were relatively bigger reductions in Eutrophication Potential and especially in Acidification Potential, mainly as a result of reduced feed protein content and subsequent nitrogen emissions from housing and manure management. The use of protease in the broiler diets reduced the environmental impacts of both feed production and broiler production. The latter is mainly through reduced ammonia emissions, which has substantial benefit per se in the poultry industry. © 2015 Society of Chemical Industry.

  10. Alkaline catalyzed biodiesel production from moringa oleifera oil with optimized production parameters

    Energy Technology Data Exchange (ETDEWEB)

    Kafuku, G.; Mbarawa, M. [Department of Mechanical Engineering, Tshwane University of Technology, Private Bag X680, 0001 Pretoria (South Africa)

    2010-08-15

    The utilization of non-edible feedstock such as moringa oleifera for biodiesel production attracts much attention owing to the issue with regards to avoiding a threat to food supplies. In this study, the optimization of biodiesel production parameters for moringa oleifera oil was carried out. The free fatty acid value of moringa oil was found to be 0.6%, rendering the one step alkaline transesterification method for converting moringa fatty acids to their methyl esters possible. The optimum production parameters: catalyst amount, alcohol amount, temperature, agitation speed and reaction time were determined experimentally and found to be: 1.0 wt% catalyst amount, 30 wt% methanol amount, 60 C reaction temperature, 400 rpm agitation rate and 60 min reaction time. With these optimal conditions the conversion efficiency was 82%. The properties of the moringa biodiesel that was produced were observed to fall within the recommended international biodiesel standards. However, moringa biodiesel showed high values of cloud and pour points of 10 C and 3 C respectively, which present a problem as regards use in cold temperatures. (author)

  11. Heterogeneous production of proteases from Brazilian clinical isolates of Pseudomonas aeruginosa.

    Science.gov (United States)

    Galdino, Anna Clara M; Viganor, Lívia; Ziccardi, Mariangela; Nunes, Ana Paula F; Dos Santos, Kátia R N; Branquinha, Marta H; Santos, André L S

    2017-12-01

    Pseudomonas aeruginosa is an important human pathogen that causes severe infections in a wide range of immunosuppressed patients. Herein, we evaluated the proteolytic profiles of 96 Brazilian clinical isolates of P. aeruginosa recovered from diverse anatomical sites. Cell-associated and extracellular proteases were evidenced by gelatin-SDS-PAGE and by the cleavage of soluble gelatin. Elastase was measured by using the peptide substrate N-succinyl-Ala-Ala-Ala-p-nitroanilide. The prevalence of elastase genes (lasA and lasB) was evaluated by PCR. Bacterial extracts were initially applied on gelatin-SDS-PAGE and the results revealed four distinct zymographic profiles as follows: profile I (composed by bands of 145, 118 and 50kDa), profile II (118 and 50kDa), profile III (145kDa) and profile IV (118kDa). All the proteolytic enzymes were inhibited by EDTA, identifying them as metalloproteases. The profile I was the most detected in both cellular (79.2%) and extracellular (84.4%) extracts. Overall, gelatinase and elastase activities measured in the spent culture media were significantly higher (around 2-fold) compared to the cellular extracts and the production level varied according to the site of bacterial isolation. For instance, tracheal secretion isolates produced elevated amount of gelatinase and elastase measured in both cellular and extracellular extracts. The prevalence of elastase genes revealed that 100% isolates were lasB-positive and 85.42% lasA-positive. Some positive/negative correlations were showed concerning the production of gelatinase, elastase, isolation site and antimicrobial susceptibility. The protease production was highly heterogeneous in Brazilian clinical isolates of P. aeruginosa, which corroborates the genomic/metabolic versatility of this pathogen. Copyright © 2016 Elsevier España, S.L.U. and Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.

  12. Biomass and biofertilizer production by Sesbania cannabina in alkaline soil

    Energy Technology Data Exchange (ETDEWEB)

    Rao, D.L.N.; Gill, H.S. [Central Soil Salinity Research Inst., Haryana (India)

    1995-12-01

    Biomass shortages in developing countries require increased investigation into fast-growing, N-fixing, woody plant species. In field trials in north India, the potential of Sesbania cannabina for production of green leaf manure (biofertilizer) and firewood (woody biomass) was investigated. At 100 days after sowing (DAS), green matter was 21.5 and 9.4 Mg ha{sup -1} in the stem and the leaf. A seeding rate of 15 kg ha{sup -1} producing a population of 10{sup 5} plants per hectare was adequate. Biofertilizer potential was 124.7 N, 5.3 P, 80.7 K and 12.0 S (kg ha{sup -1}), respectively. Nodulation was profuse and effective and N fixed was nearly 122 kg ha{sup -1} at 100 DAS. At maturity, 200 DAS, woody biomass production was 19.2 Mg ha{sup -1} and growing Sesbania until this stage was no more demanding on soil nutrients than growing it for green-matter production. There was a considerable beneficial influence from growing Sesbania on soil C and N status. (Author)

  13. Production of proteases from organic wastes by solid-state fermentation: downstream and zero waste strategies.

    Science.gov (United States)

    Marín, Maria; Artola, Adriana; Sánchez, Antoni

    2018-04-01

    Production of enzymes through solid-state fermentation (SSF) of agro-industrial wastes reports high productivity with low investment. The extraction of the final product from the solid waste and solid disposal represent the main cost of the process. In this work, the complete downstream processes of SSF of two industrial residues for the production of proteases, soy fibre (SF) and a mixture of hair and sludge (HS), were studied in terms of activity recovery, using different extraction parameters (extracting solvent, ratio solid: solvent and extraction mode). Activity after lyophilisation was tested. Solid waste valorisation after extraction was studied using respiration techniques and biogas production tests, as part of a zero waste strategy. Results showed a maximum extraction yield of 91% for SF and 121% for HS, both in agitated mode and distilled water as extraction agent. An average activity recovery of 95 ± 6 and 94 ± 6% for SF and HS, respectively, was obtained after lyophilisation and redissolution. To reduce the cost of extraction, a ratio 1:3 w : v solid-solvent in static mode is advised for SF, and 1:2 w : v extraction ratio in agitated mode for HS, both with distilled water as extracting agent. Both composting and anaerobic digestion are suitable techniques for valorisation of the waste material.

  14. Production of arapaima protein hydrolysate using Aspergillus flavo-furcatis protease and pancreatin

    Directory of Open Access Journals (Sweden)

    Flávia de Carvalho Paiva

    2015-03-01

    Full Text Available The processing of arapaima (Arapaima gigas generates a lot of residues that can be used for the development of new products of industrial interest. This study aimed at evaluating the production of protein hydrolysates from arapaima residues using Aspergillus flavo-furcatis protease and commercial pancreatin, as well as characterizing their nutritional and microbiological qualities. The raw material used was meat mechanically separated from arapaima carcasses (MMSA. Two products were developed: a protein hydrolysate of arapaima using a commercial enzyme (PHACE and another one using microbial enzyme (PHAME. The MMSA and the hydrolysates were analyzed for chemical composition, microbiological quality, degree of hydrolysis, digestibility and amino acid profile. The results showed that the PHACE protein content was 73.47 %. This value was significantly higher, when compared to the PHAME (58.03 %. However, both products showed high digestibility values, absence of microbial contaminants and reduced lipid content. Among the enzymes used, pancreatin was the most efficient one in the preparation of the final product, which showed essential amino acids content higher than the requirements for human adults. The hydrolysate developed using A. flavo-furcatis enzymes presented essential amino acids score lower than 1.0, being tryptophan the most limiting one.

  15. Hydrogen production by supercritical water gasification of alkaline black liquor

    Energy Technology Data Exchange (ETDEWEB)

    Cao, Changqing; Guo, Liejin; Chen, Yunan; Lu, Youjun [Xi' an Jiatong Univ. (China)

    2010-07-01

    Black liquor was gasified continuously in supercritical water successfully and the main gaseous products were H{sub 2}, CO{sub 2} and CH{sub 4} with little amount of CO, C{sub 2}H{sub 4} and C{sub 2}H{sub 6}. The increase of the temperature and the decrease of the flow rate and black liquor concentration enhanced SCWG of black liquor. The change of the system pressure had limited influence on the gasification effect. The maximal COD removal efficiency of 88.69 % was obtained at the temperature of 600 C. The pH values of the aqueous residue were all decreased to the range of 6.4{proportional_to}8 while the pH value of cooling effluence below 360 C increased to about 11 and the sodium content was much higher than that in the aqueous residue. The reaction rate for COD degradation in supercritical water was obtained by assuming pseudo first order reaction. And the activation energy and pre-exponential for COD removal in SCWG were 74.38kJ/mol and 1.11 x 10{sup 4} s{sup -1} respectively. (orig.)

  16. Production, purification, and characterization of an extracellular acid protease from the marine Antarctic yeast Rhodotorula mucilaginosa L7.

    Science.gov (United States)

    Lario, Luciana Daniela; Chaud, Luciana; Almeida, María das Graças; Converti, Attilio; Durães Sette, Lara; Pessoa, Adalberto

    2015-11-01

    The production, purification, and characterization of an extracellular protease released by Rhodotorula mucilaginosa L7 were evaluated in this study. This strain was isolated from an Antarctic marine alga and previously selected among others based on the capacity to produce the highest extracellular proteolytic activity in preliminary tests. R. mucilaginosa L7 was grown in Saboraud-dextrose medium at 25 °C, and the cell growth, pH of the medium, extracellular protease production and the glucose and protein consumption were determined as a function of time. The protease was then purified, and the effects of pH, temperature, and salt concentration on the catalytic activity and enzyme stability were determined. Enzyme production started at the beginning of the exponential phase of growth and reached a maximum after 48 h, which was accompanied by a decrease in the pH as well as reductions of the protein and glucose concentrations in the medium. The purified protease presented optimal catalytic activity at pH 5.0 and 50 °C. Finally, the enzyme was stable in the presence of high concentrations of NaCl. These characteristics are of interest for future studies and may lead to potential biotechnological applications that require enzyme activity and stability under acidic conditions and/or high salt concentrations. Copyright © 2015 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

  17. A Pseudomonas aeruginosa strain isolated from a contact lens-induced acute red eye (CLARE) is protease-deficient.

    Science.gov (United States)

    Estrellas, P S; Alionte, L G; Hobden, J A

    2000-03-01

    Pseudomonas aeruginosa proteases are thought to be important virulence factors in the pathogenesis of corneal disease. This study examined protease production from two strains of P. aeruginosa responsible for two very distinct clinical diseases: strain Paer1, isolated from a Contact Lens-induced Acute Red Eye (CLARE), and strain KEI 1025, isolated from a corneal ulcer. Strains were compared to a laboratory strain (ATCC 19660) known to produce severe keratitis in experimentally infected mice for protease production and for ocular virulence. Protease production was examined with colorimetric assays, gelatin zymography and western blots. Elastase A activity was quantitated with a staphylolytic assay. Ocular virulence was examined using a mouse scratch model of keratitis. In contrast to strains KEI 1025 or ATCC 19660, Paer1 was unable to produce enzymatically active elastase A, elastase, and protease IV. All three strains produced active alkaline protease. Strains KEI 1025 and ATCC 19660 produced a fulminant keratitis in mice whereas Paer1 produced a mild transient infection. Restoration of elastase activity in Paer1 via genetic complementation did not result in a virulent phenotype. Co-infection of mouse eyes with strains Paer1 and ATCC 19660 resulted in the eventual loss of Paer1 from corneal tissue. These studies suggest that P. aeruginosa elastase A and/or protease IV, but not alkaline protease or elastase, contribute to the ocular virulence of this organism.

  18. Effect of alkaline microwaving pretreatment on anaerobic digestion and biogas production of swine manure.

    Science.gov (United States)

    Yu, Tao; Deng, Yihuan; Liu, Hongyu; Yang, Chunping; Wu, Bingwen; Zeng, Guangming; Lu, Li; Nishimura, Fumitake

    2017-05-10

    Microwave assisted with alkaline (MW-A) condition was applied in the pretreatment of swine manure, and the effect of the pretreatment on anaerobic treatment and biogas production was evaluated in this study. The two main microwaving (MW) parameters, microwaving power and reaction time, were optimized for the pretreatment. Response surface methodology (RSM) was used to investigate the effect of alkaline microwaving process for manure pretreatment at various values of pH and energy input. Results showed that the manure disintegration degree was maximized of 63.91% at energy input of 54 J/g and pH of 12.0, and variance analysis indicated that pH value played a more important role in the pretreatment than in energy input. Anaerobic digestion results demonstrated that MW-A pretreatment not only significantly increased cumulative biogas production, but also shortened the duration for a stable biogas production rate. Therefore, the alkaline microwaving pretreatment could become an alternative process for effective treatment of swine manure.

  19. Cold alkaline extraction as a pretreatment for bioethanol production from eucalyptus, sugarcane bagasse and sugarcane straw

    International Nuclear Information System (INIS)

    Carvalho, Danila Morais de; Sevastyanova, Olena; Queiroz, José Humberto de; Colodette, Jorge Luiz

    2016-01-01

    Highlights: • Mathematical approach to optimize the process of cold alkaline extraction. • Hemicelluloses and lignin removal from biomasses by cold alkaline extraction. • Higher xylan and lignin removal for straw during pretreatment. • Formation of pseudo-extractives for eucalyptus during pretreatment. • Higher ethanol production for pretreated sugarcane straw. - Abstract: Optimal conditions for the cold alkaline extraction (CAE) pretreatment of eucalyptus, sugarcane bagasse and sugarcane straw are proposed in view of their subsequent bioconversion into ethanol through the semi-simultaneous saccharification and fermentation (SSSF) process (with presaccharification followed by simultaneous saccharification and fermentation, or SSF). The optimum conditions, which are identified based on an experiment with a factorial central composite design, resulted in the removal of 46%, 52% and 61% of the xylan and 15%, 37% and 45% of the lignin for eucalyptus, bagasse and straw, respectively. The formation of pseudo-extractives was observed during the CAE of eucalyptus. Despite the similar glucose concentration and yield for all biomasses after 12 h of presaccharification, the highest yield (0.065 g_e_t_h_a_n_o_l/g_b_i_o_m_a_s_s), concentrations (5.74 g L"−"1) and volumetric productivity for ethanol (0.57 g L"−"1 h"−"1) were observed for the sugarcane straw. This finding was most likely related to the improved accessibility of cellulose that resulted from the removal of the largest amount of xylan and lignin.

  20. Membrane treatment of alkaline bleaching effluents from elementary chlorine free kraft softwood cellulose production.

    Science.gov (United States)

    Oñate, Elizabeth; Rodríguez, Edgard; Bórquez, Rodrigo; Zaror, Claudio

    2015-01-01

    This paper reports experimental results on the sequential use of ultrafiltration (UF), nanofiltration (NF) and reverse osmosis (RO) to fractionate alkaline extraction bleaching effluents from kraft cellulose production. The aim was to unveil the way key pollutants are distributed when subjected to sequential UF/NF/RO membrane separation processes. Alkaline bleaching effluents were obtained from a local pinewood-based mill, featuring elementary chlorine free bleaching to produce high-brightness cellulose. The experimental system was based on a laboratory-scale membrane system, DSS LabStak® M20 Alfa Laval, using Alfa Laval UF and NF/RO membranes, operated at a constant transmembrane pressure (6 bar for UF membranes and 32 bar for NF/RO membranes), at 25°C. Results show that 78% chemical oxygen demand (COD) and total phenols, 82% adsorbable organic halogens (AOX) and 98% colour were retained by UF membranes which have molecular weight cut-off (MWCO) above 10 kDa. In all, 16% of original COD, total phenols and AOX, and the remaining 2% colour were retained by UF membranes within the 1 to 10 kDa MWCO range. Chloride ions were significantly present in all UF permeates, and RO was required to obtain a high-quality permeate with a view to water reuse. It is concluded that UF/NF/RO membranes offer a feasible option for water and chemicals recovery from alkaline bleaching effluents in kraft pulp production.

  1. Proposing and evaluating applications for products obtained during chromium chip alkaline hydrolysis produced during leather tanning

    Directory of Open Access Journals (Sweden)

    Andrea Díaz

    2006-09-01

    Full Text Available Some applications for products obtained by chromium chip alkaline hydrolysis produced during leather tanning were evaluated in this work, considering the concept of maximising tanneries’ solid residue reuse for different industrial applications and minimising the environmental impact so produced. When Cr(OH is transformed into Cr (OH(SO it can be used in tanning leather (i.e. as tanning salt. When compared to commercial salts, 2 4 it was determined that it could be applied to mixtures containing this salt, replacing it by up to 40%. Chromium content reduction was evaluated for collagen hydrolyzate by pH control after alkaline hydrolysis of the chips and by applying adsorbent materials such as bentonite, alfalfa and sorghum biomass and activated charcoal, a maximum 55% Cr removal being obtained when the first two adsorbent materials were used.

  2. Production, characterization, and immobilization of partially purified surfactant-detergent and alkali-thermostable protease from newly isolated Aeromonas caviae.

    Science.gov (United States)

    Datta, Sumitra; Menon, Gopalakrishnan; Varughese, Bincy

    2017-04-21

    Proteolytic Aeromonas caviae P-1-1 growing at wide-ranging pH (7.0-11.0) and moderate salinity (0-5% NaCl) was isolated from cattle shed of Thanjavur, India. It produced lipase, gelatinase, and polyhydroxybutyrate. Different culture conditions, incubation time, carbon and nitrogen sources, vitamins, amino acids, surfactants, and metal ions for optimal growth and protease production of P-1-1 were examined. Maximum protease (0.128 U/mL) production was achieved with 1% fructose, 1% yeast extract, 0.1% ammonium sulfate, 3% NaCl, 0.1% CaCl 2  · 2H 2 O, 1% glycine, 0.1% vitamin E, and 0.1% Tween-40 at pH 8.0 after 42 hr of incubation at 37°C. It was active over broad range of pH (7.0-12.0), temperature (15-100°C), and salinity (0-9% NaCl) with optima at pH 10.0, 55°C, and 3% NaCl. It retained 65 and 48% activities at pH 12.0 and 100°C, respectively. Partially purified protease was highly stable (100%) within pH range 7.0-12.0 and salinities of 0-5% NaCl for 48 hr. Cu 2+ , Mn 2+ , Co 2+ , and Ca 2+ did not inhibit its activity. Its stability at extreme pHs, temperatures, and in the presence of surfactants and commercial detergents suggests its possible application in laundry detergents. Partially purified protease was immobilized and reused. This is the first report of alkali-thermotolerant, surfactant-detergent-stable partially purified extracellular protease from A. caviae.

  3. PhAP protease from Pseudoalteromonas haloplanktis TAC125: Gene cloning, recombinant production in E. coli and enzyme characterization

    Science.gov (United States)

    de Pascale, D.; Giuliani, M.; De Santi, C.; Bergamasco, N.; Amoresano, A.; Carpentieri, A.; Parrilli, E.; Tutino, M. L.

    2010-08-01

    Cold-adapted proteases have been found to be the dominant activity throughout the cold marine environment, indicating their importance in bacterial acquisition of nitrogen-rich complex organic compounds. However, few extracellular proteases from marine organisms have been characterized so far, and the mechanisms that enable their activity in situ are still largely unknown. Aside from their ecological importance and use as model enzyme for structure/function investigations, cold-active proteolytic enzymes offer great potential for biotechnological applications. Our studies on cold adapted proteases were performed on exo-enzyme produced by the Antarctic marine bacterium Pseudoalteromonas haloplanktis TAC125. By applying a proteomic approach, we identified several proteolytic activities from its culture supernatant. PhAP protease was selected for further investigations. The encoding gene was cloned and the protein was recombinantly produced in E. coli cells. The homogeneous product was biochemically characterised and it turned out that the enzyme is a Zn-dependent aminopeptidase, with an activity dependence from assay temperature typical of psychrophilic enzymes.

  4. Effect of temperature, pH and metal lons on the activity and stability of alkaline protease from novel bacillus licheniformis mzk03

    International Nuclear Information System (INIS)

    Sayem, S.M.A.; Hoq, M.M.; Alam, M.J.

    2006-01-01

    The effect of temperature, pH and metal ions on the activity and stability of crude protease from Bacillus licheniformis MZK03 was studied. The fermentation in shake culture revealed that maximum level of enzyme was produced at 37 degree C and pH 8.5 after 39 hr at 120 rpm. It lost its activity rapidly above 50 degree C and half-life of the protease at this temperature was 50 min with optimum activity at 40 degree C. It was most stable at pH 8.5 and lost its activity rapidly above pH 10.0, and at pH 11.0 reached 30% of the activity obtained at pH 9.0. The enzyme lost its activity completely at pH 13.0. Optimum proteolytic activity was found at 40 degree C and pH 9.5. The enzyme activity was accelerated by the addition of Mg/sup 2+/, Ca/sup 2+/ and Mn/sup 2+/, whereas it was inhibited by Hg/sup 2+/. (author)

  5. Production of lactic acid from C6-polyols by alkaline hydrothermal reactions

    International Nuclear Information System (INIS)

    Zhou Huazhen; Jin Fangming; Wu Bing; Cao Jianglin; Duan Xiaokun; Kishita, Atsushi

    2010-01-01

    Production of lactic acid from C6-polyols (Mannitol) under alkaline hydrothermal conditions was investigated. Experiments were performed to examine the difference in the production of lactic acid between C6-polyols and C3-polyols (glycerine), as well as C6-aldoses (glucose). Results showed that the yield of lactic acid from C6-polyols was lower than that from both glycerine and glucose. It indicated that long chain polyols might follow a different reaction pathway from that of glycerine. Further investigation is needed to clarify the reaction mechanism and improve the relatively low lactic acid acid yield from C6-polyols.

  6. A functional genomics study of extracellular protease production by Aspergillus niger

    OpenAIRE

    Braaksma, Machtelt

    2010-01-01

    The objective of the project described in this thesis was to study the complex induction of extracellular proteases in the filamentous fungus Aspergillus niger using information gathered with functional genomics technologies. A special emphasis is given to the requirements for performing a successful systems biology study and addressing the challenges met in analyzing the large, information-rich data sets generated with functional genomics technologies. The role that protease activity plays i...

  7. Effect of sorghum type and malting on production of free amino nitrogen in conjunction with exogenous protease enzymes.

    Science.gov (United States)

    Dlamini, Bhekisisa C; Buys, Elna M; Taylor, John R N

    2015-01-01

    Sorghum types suitable for brewing and bioethanol production are required. The effect of sorghum type (white non-tannin versus white type II tannin) on free amino nitrogen (FAN) production from sorghum grain and malt using exogenous protease enzymes was investigated over extended incubation at moderate temperature (45 °C). With grain in the absence of exogenous proteases, white non-tannin sorghum produced substantially higher levels of FAN than white type II tannin sorghum, due to the tannins in the latter. Incubating sorghum grain with neutral proteinase and amino-peptidase in combination improved FAN production. The two sorghum types produced similar FAN levels when malted and incubated in the absence of the exogenous proteases. When both sorghums were malted and incubated with neutral proteinase alone substantially more FAN yield (124-126 mg 100 g(-1)) occurred than with grains (61-84 mg 100 g(-1)). The combination of amino-peptidase and proteinase did not improve FAN further. Neither, did malting influence wort free amino acid profile. Group B amino acids constituted the highest percentage (42-47%). With grain, white non-tannin sorghum plus proteinase and amino-peptidase yields the highest FAN, with malt both white non-tannin and white type II tannin sorghums plus proteinase yield the highest FAN. © 2014 Society of Chemical Industry.

  8. Purification and Biochemical Characterization of a Neutral Serine Protease from Trichoderma harzianum. Use in Antibacterial Peptide Production from a Fish By-Product Hydrolysate.

    Science.gov (United States)

    Aissaoui, Neyssene; Chobert, Jean-Marc; Haertlé, Thomas; Marzouki, M Nejib; Abidi, Ferid

    2017-06-01

    This study reports the purification and biochemical characterization of an extracellular neutral protease from the fungus Trichoderma harzianum. The protease (Th-Protease) was purified from the culture supernatant to homogeneity by a three-step procedure with 14.2% recovery and 9.06-fold increase in specific activity. The purified enzyme appeared as a single protein band after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with a molecular mass of about 20 kDa. The optimum pH and temperature for the proteolytic activity were pH 7.0 and 40 °C, respectively. The enzyme was then investigated for its potential application in the production of antibacterial peptides. Interestingly, Scorpaena notata viscera protein hydrolysate prepared using the purified serine protease (Th-Protease) showed remarkable in vitro antibacterial activities. A peptide with a high antibacterial activity was further purified by a three-step procedure, and its sequence was identified as FPIGMGHGSRPA. The result of this study offers a promising alternative to produce natural antibacterial peptides from fish protein hydrolysate.

  9. Geochemical modeling of the influence of silicate mineral alteration on alkalinity production and carbonate precipitation

    Science.gov (United States)

    Herda, Gerhard; Kraemer, Stephan M.; Gier, Susanne; Meister, Patrick

    2016-04-01

    High CO2 partial pressure (pCO2) in deep rock reservoirs causes acidification of the porefluid. Such conditions occur during injection and subsurface storage of CO2 (to prevent the release of greenhouse gas) but also naturally in zones of strong methanogenic microbial activity in organic matter-rich ocean margin sediments. The acidic fluids are corrosive to carbonates and bear the risk of leakage of CO2 gas to the surface. Porefluid acidification may be moderated by processes that increase the alkalinity, i.e. that produce weak acid anions capable of buffering the acidification imposed by the CO2. Often, alkalinity increases as a result of anaerobic microbial activity, such as anaerobic oxidation of methane. However, on a long term the alteration of silicates, in particular, clay minerals, may be a more efficient mechanism of alkalinity production. Under altered temperature, pressure and porefluid composition at depth, clay minerals may change to thermodynamically more stable states, thereby increasing the alkalinity of the porefluid by partial leaching of Mg-(OH)2 and Ca-(OH)2 (e.g. Wallmann et al., 2008; Mavromatis et al., 2014). This alteration may even be enhanced by a high pCO2. Thus, silicate alteration can be essential for a long-term stabilization of volatile CO2 in the form of bicarbonate or may even induce precipitation of carbonate minerals, but these processes are not fully understood yet. The goal of this study is to simulate the alkalinity effect of silicate alteration under diagenetic conditions and high pCO2 by geochemical modeling. We are using the program PHREEQC (Parkhurst and Appelo, 2013) to generate high rock/fluid ratio characteristics for deep subsurface rock reservoirs. Since we are interested in the long-term evolution of diagenetic processes, over millions of years, we do not consider kinetics but calculate the theoretically possible equilibrium conditions. In a first step we are calculating the saturation state of different clay minerals

  10. Fuelwood production potential of six Prosopis species on an alkaline soil site

    Energy Technology Data Exchange (ETDEWEB)

    Goel, V.L.; Behl, H.M. [National Botanical Research Inst., Lucknow (India). Biomass Research Center

    1995-07-01

    The biomass potential of six species Prosopis was evaluated on highly alkaline soil site. Prosopis alba I was found to have the fastest growth rate and highest above-ground biomass production. P. juliflora ranked next. P. cineraria showed high plant establishment but relatively slow growth. The performance of P. glandulosa was poor on such sites. The high fuelwood value index and rapid growth rate of P. juliflora and P. alba makes them suitable for short-rotation fuelwood forestry programmes on waste-lands. Selection of promising genotypes is suggested as a means of improvement in yields. (author)

  11. Activated factor X signaling via protease-activated receptor 2 suppresses pro-inflammatory cytokine production from LPS-stimulated myeloid cells.

    LENUS (Irish Health Repository)

    Gleeson, Eimear M

    2013-07-19

    Vitamin K-dependent proteases generated in response to vascular injury and infection enable fibrin clot formation, but also trigger distinct immuno-regulatory signaling pathways on myeloid cells. Factor Xa, a protease crucial for blood coagulation, also induces protease-activated receptor-dependent cell signaling. Factor Xa can bind both monocytes and macrophages, but whether factor Xa-dependent signaling stimulates or suppresses myeloid cell cytokine production in response to Toll-like receptor activation is not known. In this study, exposure to factor Xa significantly impaired pro-inflammatory cytokine production from lipopolysaccharide-treated peripheral blood mononuclear cells, THP-1 monocytic cells and murine macrophages. Furthermore, factor Xa inhibited nuclear factor-kappa B activation in THP-1 reporter cells, requiring phosphatidylinositide 3-kinase activity for its anti-inflammatory effect. Active-site blockade, γ-carboxyglutamic acid domain truncation and a peptide mimic of the factor Xa inter-epidermal growth factor-like region prevented factor Xa inhibition of lipopolysaccharide-induced tumour necrosis factor-α release. In addition, factor Xa anti-inflammatory activity was markedly attenuated by the presence of an antagonist of protease-activated receptor 2, but not protease-activated receptor 1. The key role of protease-activated receptor 2 in eliciting factor Xa-dependent anti-inflammatory signaling on macrophages was further underscored by the inability of factor Xa to mediate inhibition of tumour necrosis factor-α and interleukin-6 release from murine bone marrow-derived protease-activated receptor 2-deficient macrophages. We also show for the first time that, in addition to protease-activated receptor 2, factor Xa requires a receptor-associated protein-sensitive low-density lipoprotein receptor to inhibit lipopolysaccharide-induced cytokine production. Collectively, this study supports a novel function for factor Xa as an endogenous, receptor

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

    Directory of Open Access Journals (Sweden)

    Salleh Abu

    2009-04-01

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

  13. 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.

  14. DksA-HapR-RpoS axis regulates haemagglutinin protease production in Vibrio cholerae.

    Science.gov (United States)

    Basu, Pallabi; Pal, Ritesh Ranjan; Dasgupta, Shreya; Bhadra, Rupak K

    2017-06-01

    DksA acts as a co-factor for the intracellular small signalling molecule ppGpp during the stringent response. We recently reported that the expression of the haemagglutinin protease (HAP), which is needed for shedding of the cholera pathogen Vibrio cholerae during the late phase of infection, is significantly downregulated in V. cholerae ∆dksA mutant (∆dksAVc) cells. So far, it has been shown that HAP production by V. cholerae cells is critically regulated by HapR and also by RpoS. Here, we provide evidence that V. cholerae DksA (DksAVc) positively regulates HapR at both the transcriptional and post-transcriptional levels. We show that in ∆dksAVc cells the CsrB/C/D sRNAs, required for the maintenance of intracellular levels of hapR transcripts during the stationary growth, are distinctly downregulated. Moreover, the expression of exponential phase regulatory protein Fis, a known negative regulator of HapR, was found to continue even during the stationary phase in ∆dksAVc cells compared to that of wild-type strain, suggesting another layer of complex regulation of HapR by DksAVc. Extensive reporter construct-based and quantitative reverse-transcriptase PCR (qRT-PCR) analyses supported that RpoS is distinctly downregulated at the post-transcriptional/translational levels in stationary phase-grown ∆dksAVc cells. Since HAP expression through HapR and RpoS is stationary phase-specific in V. cholerae, it appears that DksAVc is also a critical stationary phase regulator for fine tuning of the expression of HAP. Moreover, experimental evidence provided in this study clearly supports that DksAVc is sitting at the top of the hierarchy of regulation of expression of HAP in V. cholerae.

  15. RE/H2 Production Micro-System Based on Standard Alkaline Electrolytic Technology

    International Nuclear Information System (INIS)

    Moschetto, A.; Tina, G.M.; Ferraro, M.; Briguglio, N.; Antonucci, V.

    2006-01-01

    This paper presents the first task of a more comprehensive research project focused on the development of micro-scale (1-20 kW) Renewable Hydrogen (RE/H 2 ) production systems oriented to carry on a wide campaign of educational and demonstration projects. The paper proposes to rely on low-cost and rugged 'standard' alkaline electrolytic technology, well suited for decentralized hydrogen production, but requiring a certain R and D effort to get technical competitiveness. An electrolyser test facility has been designed and carried out. Then performance assessment of a commercial electrolyser and its sub-systems has been accomplished. First experimental results stated that the unit under test gets an average production efficiency of 51%, versus a stack (cell) efficiency of about 62%, while the aged AC/DC power converter, to be removed or replaced to adapt the unit to DC link with renewables, requires more than 16% of the incoming power. (authors)

  16. RE/H{sub 2} Production Micro-System Based on Standard Alkaline Electrolytic Technology

    Energy Technology Data Exchange (ETDEWEB)

    Moschetto, A.; Tina, G.M. [UNICT DIEES University of Catania - Electric, Electronic and Systemcs Department, Viale A. Doria, 5 - 95125 Catania, (Italy); Ferraro, M.; Briguglio, N.; Antonucci, V. [CNR ITAE, National Council of Research - Advanced Energy Technology Institute, Via Salita S. Lucia, 5 - 98128 Messina, (Italy)

    2006-07-01

    This paper presents the first task of a more comprehensive research project focused on the development of micro-scale (1-20 kW) Renewable Hydrogen (RE/H{sub 2}) production systems oriented to carry on a wide campaign of educational and demonstration projects. The paper proposes to rely on low-cost and rugged 'standard' alkaline electrolytic technology, well suited for decentralized hydrogen production, but requiring a certain R and D effort to get technical competitiveness. An electrolyser test facility has been designed and carried out. Then performance assessment of a commercial electrolyser and its sub-systems has been accomplished. First experimental results stated that the unit under test gets an average production efficiency of 51%, versus a stack (cell) efficiency of about 62%, while the aged AC/DC power converter, to be removed or replaced to adapt the unit to DC link with renewables, requires more than 16% of the incoming power. (authors)

  17. Anaerobic digestion of the microalga Spirulina at extreme alkaline conditions: biogas production, metagenome, and metatranscriptome

    Science.gov (United States)

    Nolla-Ardèvol, Vímac; Strous, Marc; Tegetmeyer, Halina E.

    2015-01-01

    A haloalkaline anaerobic microbial community obtained from soda lake sediments was used to inoculate anaerobic reactors for the production of methane rich biogas. The microalga Spirulina was successfully digested by the haloalkaline microbial consortium at alkaline conditions (pH 10, 2.0 M Na+). Continuous biogas production was observed and the obtained biogas was rich in methane, up to 96%. Alkaline medium acted as a CO2 scrubber which resulted in low amounts of CO2 and no traces of H2S in the produced biogas. A hydraulic retention time (HRT) of 15 days and 0.25 g Spirulina L−1 day−1 organic loading rate (OLR) were identified as the optimal operational parameters. Metagenomic and metatranscriptomic analysis showed that the hydrolysis of the supplied substrate was mainly carried out by Bacteroidetes of the “ML635J-40 aquatic group” while the hydrogenotrophic pathway was the main producer of methane in a methanogenic community dominated by Methanocalculus. PMID:26157422

  18. A functional genomics study of extracellular protease production by Aspergillus niger

    NARCIS (Netherlands)

    Braaksma, Machtelt

    2010-01-01

    The objective of the project described in this thesis was to study the complex induction of extracellular proteases in the filamentous fungus Aspergillus niger using information gathered with functional genomics technologies. A special emphasis is given to the requirements for performing a

  19. Soybean hull induced production of carbohydrases and protease among Aspergillus and their effectiveness in soy flour carbohydrate and protein separation.

    Science.gov (United States)

    Li, Qian; Loman, Abdullah Al; Coffman, Anthony M; Ju, Lu-Kwang

    2017-04-20

    Soybean hull consists mainly of three major plant carbohydrates, i.e., cellulose, hemicellulose and pectin. It is inexpensive and a good potential substrate for carbohydrase production because it is capable of inducing a complete spectrum of activities to hydrolyze complex biomass. Aspergillus is known for carbohydrase production but no studies have evaluated and compared, among Aspergillus species and strains, the soybean hull induced production of various carbohydrases. In this study, A. aculeatus, A. cinnamomeus, A. foetidus, A. phoenicis and 11 A. niger strains were examined together with T. reesei Rut C30, another known carbohydrase producer. The carbohydrases evaluated included pectinase, polygalacturonase, xylanase, cellulase, α-galactosidase and sucrase. Growth morphology and pH profiles were also followed. Among Aspergillus strains, morphology was found to correlate with both carbohydrase production and pH decrease profile. Filamentous strains gave higher carbohydrase production while causing slower pH decrease. The enzyme broths produced were also tested for separation of soy flour carbohydrate and protein. Defatted soy flour contains about 53% protein and 32% carbohydrate. The enzymatic treatment can increase protein content and remove indigestible oligo-/poly-saccharides, and improve use of soy flour in feed and food. Protease production by different strains was therefore also compared for minimizing protein degradation. A. niger NRRL 322 and A. foetidus NRRL 341 were found to be the most potent strains that produced maximal carbohydrases and minimal protease under soybean hull induction. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Production, secretion, and stability of human secreted alkaline phosphatase in tobacco NT1 cell suspension cultures.

    Science.gov (United States)

    Becerra-Arteaga, Alejandro; Mason, Hugh S; Shuler, Michael L

    2006-01-01

    Tobacco NT1 cell suspension cultures secreting active human secreted alkaline phosphatase (SEAP) were generated for the first time as a model system to study recombinant protein production, secretion, and stability in plant cell cultures. The SEAP gene encodes a secreted form of the human placental alkaline phosphatase (PLAP). During batch culture, the highest level of active SEAP in the culture medium (0.4 U/mL, corresponding to approximately 27 mg/L) was observed at the end of the exponential growth phase. Although the level of active SEAP decreased during the stationary phase, the activity loss did not appear to be due to SEAP degradation (based on Western blots) but due to SEAP denaturation. The protein-stabilizing agents polyvinylpirrolidone (PVP) and bacitracin were added extracellularly to test for their ability to reduce the loss of SEAP activity during the stationary phase. Bacitracin (100 mg/L) was the most effective treatment at sustaining activity levels for up to 17 days post-subculture. Commercially available human placental alkaline phosphatase (PLAP) was used to probe the mechanism of SEAP deactivation. Experiments with PLAP in sterile and conditioned medium corroborated the denaturation of SEAP by factors generated by cell growth and not due to simple proteolysis. We also show for the first time that the factors promoting activity loss are heat labile at 95 degrees C but not at 70 degrees C, and they are not inactivated after a 5 day incubation period under normal culture conditions (27 degrees C). In addition, there were no significant changes in pH or redox potential when comparing sterile and cell-free conditioned medium during PLAP incubation, indicating that these factors were unimportant.

  1. A Comparison between Lime and Alkaline Hydrogen Peroxide Pretreatments of Sugarcane Bagasse for Ethanol Production

    Science.gov (United States)

    Rabelo, Sarita C.; Filho, Rubens Maciel; Costa, Aline C.

    Pretreatment procedures of sugarcane bagasse with lime (calcium hydroxide) or alkaline hydrogen peroxide were evaluated and compared. Analyses were performed using 2 × 2 × 2 factorial designs, with pretreatment time, temperature, and lime loading and hydrogen peroxide concentration as factors. The responses evaluated were the yield of total reducing sugars (TRS) and glucose released from pretreated bagasse after enzymatic hydrolysis. Experiments were performed using the bagasse as it comes from an alcohol/ sugar factory and bagasse in the size range of 0.248 to 1.397 mm (12-60 mesh). The results show that when hexoses and pentoses are of interest, lime should be the pretreatment agent chosen, as high TRS yields are obtained for nonscreened bagasse using 0.40 g lime/g dry biomass at 70 °C for 36 h. When the product of interest is glucose, the best results were obtained with lime pretreatment of screened bagasse. However, the results for alkaline peroxide and lime pretreatments of nonscreened bagasse are not very different.

  2. Aromatic products from reaction of lignin model compounds with UV-alkaline peroxide

    International Nuclear Information System (INIS)

    Sun, Y.P.; Wallis, A.F.A.; Nguyen, K.L.

    1997-01-01

    A series of guaiacyl and syringyl lignin model compounds and their methylated analogues were reacted with alkaline hydrogen peroxide while irradiating with UV light at 254 nm. The aromatic products obtained were investigated by gas chromatography-mass spectrometry (GC-MS). Guaiacol, syringol and veratrol gave no detectable aromatic products. However, syringol methyl ether gave small amounts of aromatic products, resulting from ring substitution and methoxyl displacement by hydroxyl radicals. Reaction of vanillin and syringaldehyde gave the Dakin reaction products, methoxy-1,4-hydroquinones, while reaction of their methyl ethers yielded benzoic acids. Acetoguaiacone, acetosyringone and their methyl ethers afforded several hydroxylated aromatic products, but no aromatic products were identified in the reaction mixtures from guaiacylpropane and syringylpropane. In contrast, veratrylpropane gave a mixture from which 17 aromatic hydroxylated compounds were identified. It is concluded that for phenolic lignin model compounds, particularly those possessing electrondonating aromatic ring substituents, ring-cleavage reactions involving superoxide radical anions are dominant, whereas for non-phenolic lignin models, hydroxylation reactions through attack of hydroxyl radicals prevail

  3. Production of protease and lipase by solvent tolerant Pseudomonas aeruginosa PseA in solid-state fermentation using Jatropha curcas seed cake as substrate.

    Science.gov (United States)

    Mahanta, Nilkamal; Gupta, Anshu; Khare, S K

    2008-04-01

    Deoiled Jatropha seed cake was assessed for its suitability as substrate for enzyme production by solid-state fermentation (SSF). Solvent tolerant Pseudomonas aeruginosa PseA strain previously reported by us was used for fermentation. The seed cake supported good bacterial growth and enzyme production (protease, 1818 U/g of substrate and lipase, 625 U/g of substrate) as evident by its chemical composition. Maximum protease and lipase production was observed at 50% substrate moisture, a growth period of 72 and 120 h, and a substrate pH of 6.0 and 7.0, respectively. Enrichment with maltose as carbon source increased protease and lipase production by 6.3- and 1.6-fold, respectively. Nitrogen supplementation with peptone for protease and NaNO(3) for lipase production also enhanced the enzyme yield reaching 11,376 U protease activity and 1084 U lipase activity per gram of Jatropha seed cake. These results demonstrated viable approach for utilization of this huge biomass by solid-state fermentation for the production of industrial enzymes. This offers significant benefit due to low cost and abundant availability of cake during biodiesel production.

  4. Cowpea Mosaic Virus-Encoded Protease Does Not Recognize Primary Translation Products of M RNAs from Other Comoviruses

    OpenAIRE

    Goldbach, Rob; Krijt, Jette

    1982-01-01

    The protease encoded by the large (B) RNA segment of cowpea mosaic virus was tested for its ability to recognize the in vitro translation products of the small (M) RNA segment from the comoviruses squash mosaic virus, red clover mottle virus, and cowpea severe mosaic virus (CPsMV, strains Dg and Ark), and from the nepovirus tomato black ring virus. Like M RNA from cowpea mosaic virus, the M RNAs from squash mosaic virus, red clover mottle virus, CPsMV-Dg, and CPsMV-Ark were all translated int...

  5. Effectiveness of sal deoiled seed cake as an inducer for protease production from Aeromonas sp. S1 for its application in kitchen wastewater treatment.

    Science.gov (United States)

    Saini, Vandana; Bhattacharya, Amrik; Gupta, Anshu

    2013-08-01

    The present study is an attempt to demonstrate the feasibility of sal (Shorea robusta) deoiled cake--a forest-based industrial by-product--as a cheaper media supplement for augmented protease production from Aeromonas sp. S1 and application of protease in the treatment of kitchen wastewater. Under optimized conditions, protease production could successfully be enhanced to 5.13-fold (527.5 U mL(-1)) on using sal deoiled seed cake extract (SDOCE), as medium additive, compared to an initial production of 102.7 U mL(-1) in its absence. The culture parameters for optimum production of protease were determined to be incubation time (48 h), pH (7.0), SDOCE concentration (3 % (v/v)), inoculum size (0.3-0.6 % (v/v)), and agitation rate (100 rpm). The enzyme was found to have an optimum pH and temperature of 8.0 and 60 °C, respectively. The protease preparation was tested for treatment of organic-laden kitchen wastewater. After 96 h of wastewater treatment under static condition, enzyme preparation was able to reduce 74 % biological oxygen demand, 37 % total suspended solids, and 41 % oil and grease. The higher and improved level of protease obtained using sal deoiled seed cake-based media hence offers a new approach for value addition to this underutilized biomass through industrial enzyme production. The protease produced using this biomass could also be used as pretreatment tool for remediation of organic-rich food wastewater.

  6. Mathematical modeling of lipase and protease production by Penicillium restrictum in a batch fermenter.

    Science.gov (United States)

    Freire, D M; Sant'Anna, G L; Alves, T L

    1999-01-01

    This work presents a mathematical model that describes time course variations of extracellular lipase and protease activities for the batch fermentation of the fungus Penicillium restrictum, a new and promising strain isolated from soil and wastes of a Brazilian babassu coconut oil industry. The fermentation process was modeled by an unstructured model, which considered the following dependent variables: cells, fat acid, dissolved oxygen concentrations, lipase and protease activities, and cell lysate concentration. The last variable represents the amount of cells that has been lysed by the shear stress and natural cell death. Proteases released to the medium, as consequence of this process, enhance lipase inactivation. The model is able to predict the effects of some operation variables such as air flow rate and agitation speed. The mathematical model was validated against batch-fermentation data obtained under several operating conditions. Because substrate concentration has antagonistic effects on lipase activity, a typical optimization scheme should be developed in order to minimize these deleterious effects while maximizing lipase activity.

  7. Characterization and Preparation of Broken Rice Proteins Modified by Proteases

    Directory of Open Access Journals (Sweden)

    Lixia Hou

    2010-01-01

    Full Text Available Broken rice is an underutilized by-product of milling. Proteins prepared from broken rice by treatments with alkaline protease and papain have been characterized with regard to nutritional and functional properties. The protein content and the protein recovery were 56.45 and 75.45 % for alkaline protease treatment, and 65.45 and 46.32 % for papain treatment, respectively. Protease treatment increased the lysine and valine content, leading to a more balanced amino acid profile. Broken rice proteins had high emulsifying capacity, 58.3–71.6 % at neutral pH, and adequate water holding capacity, ranging from 1.96 to 2.93 g/g of proteins. At pH=7.0, the broken rice protein had the highest water holding capacity and the best interfacial activities (emulsifying capacity, emulsifying stability, foaming capacity and foaming stability, which may be the result of the higher solubility at pH=7.0. The interfacial activities increased with the increase in the mass fraction of broken rice proteins. The proteins prepared by the papain treatment had higher water holding capacity (p>0.05, emulsifying capacity (p0.05 than alkaline protease treatment at the same pH or mass fraction. To test the fortification of food products with broken rice proteins, pork sausages containing the proteins were prepared. Higher yield of the sausages was obtained with the increased content of broken rice proteins, in the range of 2.0–9.0 %. The results indicate that broken rice proteins have potential to be used as the protein fortification ingredient for food products.

  8. Optimizing HIV-1 protease production in Escherichia coli as fusion protein

    Directory of Open Access Journals (Sweden)

    Piubelli Luciano

    2011-06-01

    Full Text Available Abstract Background Human immunodeficiency virus (HIV is the etiological agent in AIDS and related diseases. The aspartyl protease encoded by the 5' portion of the pol gene is responsible for proteolytic processing of the gag-pol polyprotein precursor to yield the mature capsid protein and the reverse transcriptase and integrase enzymes. The HIV protease (HIV-1Pr is considered an attractive target for designing inhibitors which could be used to tackle AIDS and therefore it is still the object of a number of investigations. Results A recombinant human immunodeficiency virus type 1 protease (HIV-1Pr was overexpressed in Escherichia coli cells as a fusion protein with bacterial periplasmic protein dithiol oxidase (DsbA or glutathione S-transferase (GST, also containing a six-histidine tag sequence. Protein expression was optimized by designing a suitable HIV-1Pr cDNA (for E. coli expression and to avoid autoproteolysis and by screening six different E. coli strains and five growth media. The best expression yields were achieved in E. coli BL21-Codon Plus(DE3-RIL host and in TB or M9 medium to which 1% (w/v glucose was added to minimize basal expression. Among the different parameters assayed, the presence of a buffer system (based on phosphate salts and a growth temperature of 37°C after adding IPTG played the main role in enhancing protease expression (up to 10 mg of chimeric DsbA:HIV-1Pr/L fermentation broth. GST:HIVPr was in part (50% produced as soluble protein while the overexpressed DsbA:HIV-1Pr chimeric protein largely accumulated in inclusion bodies as unprocessed fusion protein. A simple refolding procedure was developed on HiTrap Chelating column that yielded a refolded DsbA:HIV-1Pr with a > 80% recovery. Finally, enterokinase digestion of resolubilized DsbA:HIV-1Pr gave more than 2 mg of HIV-1Pr per liter of fermentation broth with a purity ≤ 80%, while PreScission protease cleavage of soluble GST:HIVPr yielded ~ 0.15 mg of pure HIV-1

  9. Cowpea Mosaic Virus-Encoded Protease Does Not Recognize Primary Translation Products of M RNAs from Other Comoviruses.

    Science.gov (United States)

    Goldbach, R; Krijt, J

    1982-09-01

    The protease encoded by the large (B) RNA segment of cowpea mosaic virus was tested for its ability to recognize the in vitro translation products of the small (M) RNA segment from the comoviruses squash mosaic virus, red clover mottle virus, and cowpea severe mosaic virus (CPsMV, strains Dg and Ark), and from the nepovirus tomato black ring virus. Like M RNA from cowpea mosaic virus, the M RNAs from squash mosaic virus, red clover mottle virus, CPsMV-Dg, and CPsMV-Ark were all translated into two large polypeptides with apparent molecular weights which were different for each virus and even for the two CPsMV strains. Neither the in vitro products from squash mosaic virus, red clover mottle virus, and CPsMV M RNAs nor the in vitro product from tomato black ring virus RNA-2 were processed by the cowpea mosaic virus-encoded protease, indicating that the activity of this enzyme is highly specific.

  10. Site-Dependent Environmental Impacts of Industrial Hydrogen Production by Alkaline Water Electrolysis

    Directory of Open Access Journals (Sweden)

    Jan Christian Koj

    2017-06-01

    Full Text Available Industrial hydrogen production via alkaline water electrolysis (AEL is a mature hydrogen production method. One argument in favor of AEL when supplied with renewable energy is its environmental superiority against conventional fossil-based hydrogen production. However, today electricity from the national grid is widely utilized for industrial applications of AEL. Also, the ban on asbestos membranes led to a change in performance patterns, making a detailed assessment necessary. This study presents a comparative Life Cycle Assessment (LCA using the GaBi software (version 6.115, thinkstep, Leinfelden-Echterdingen, Germany, revealing inventory data and environmental impacts for industrial hydrogen production by latest AELs (6 MW, Zirfon membranes in three different countries (Austria, Germany and Spain with corresponding grid mixes. The results confirm the dependence of most environmental effects from the operation phase and specifically the site-dependent electricity mix. Construction of system components and the replacement of cell stacks make a minor contribution. At present, considering the three countries, AEL can be operated in the most environmentally friendly fashion in Austria. Concerning the construction of AEL plants the materials nickel and polytetrafluoroethylene in particular, used for cell manufacturing, revealed significant contributions to the environmental burden.

  11. The effect of delignification process with alkaline peroxide on lactic acid production from furfural residues

    Directory of Open Access Journals (Sweden)

    Yong Tang

    2012-11-01

    Full Text Available Furfural residues produced from the furfural industry were investigated as a substrate for lactic acid production by simultaneous saccharification and fermentation (SSF. Alkaline peroxide was used for delignification of furfural residues to improve the final lactic acid concentration. The residue was treated with 1.3% to 1.7% hydrogen peroxide at 80 °C for 1 h with a substrate concentration of 3.33%. SSF of furfural residues with different delignification degrees were carried out to evaluate the effect of delignification degree on lactic acid production. Using corn hydrolysates/ furfural residues as substrates, SSF with different media were carried out to investigate the effect of lignin on the interaction between enzymes and lactic acid bacteria. Lactic acid bacteria had a negative effect on cellulase, thus resulting in the reduction of enzyme activity. Lignin and nutrients slowed down the decreasing trend of enzyme activity. A higher delignification resulted in a slower fermentation rate and lower yield due to degradation products of lignin and the effect of lignin on the interaction between enzymes and lactic acid bacteria. For the purpose of lactic acid production, a moderate delignification (furfural residues with the lignin content of 14.8% was optimum.

  12. Simultaneous production of detergent stable keratinolytic protease, amylase and biosurfactant by Bacillus subtilis PF1 using agro industrial waste.

    Science.gov (United States)

    Bhange, Khushboo; Chaturvedi, Venkatesh; Bhatt, Renu

    2016-06-01

    The present study is an attempt to optimize simultaneous production of keratinolytic protease, amylase and biosurfactant from feather meal, potato peel and rape seed cake in a single media by response surface methodology to evaluate their biochemical properties for detergent additive. The optimization was carried out using 20 run, 3 factor and 5-level of central composite design on design expert software which resulted in a 1.2, 0.84 and 2.28 fold increase in protease, amylase and biosurfactant production. The proteolytic activity was found to be optimum at pH 9.0 and 60 °C while optimum amylolytic activity was recorded at pH 6.0 and 70 °C respectively. Both enzymes were found to be stable in the presence of organic solvents, ionic and commercial detergent and oxidizing agents. The biosurfactant was extracted with chloroform and was found to be stable at varying pH and temperature; however a reduction in the activity was observed at temperature higher than 70 °C. The isolated enzymes and biosurfactants may find applications in the effective removal of stains.

  13. Enhanced enzymatic hydrolysis and ethanol production from cashew apple bagasse pretreated with alkaline hydrogen peroxide.

    Science.gov (United States)

    da Costa, Jessyca Aline; Marques, José Edvan; Gonçalves, Luciana Rocha Barros; Rocha, Maria Valderez Ponte

    2015-03-01

    The effect of combinations and ratios between different enzymes has been investigated in order to assess the optimal conditions for hydrolysis of cashew apple bagasse pretreated with alkaline hydrogen peroxide (the solids named CAB-AHP). The separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) processes were evaluated in the ethanol production. The enzymatic hydrolysis conducted with cellulase complex and β-glucosidase in a ratio of 0.61:0.39, enzyme loading of 30FPU/g(CAB-AHP) and 66CBU/g(CAB-AHP), respectively, using 4% cellulose from CAB-AHP, turned out to be the most effective conditions, with glucose and xylose yields of 511.68 mg/g(CAB-AHP) and 237.8 mg/g(CAB-AHP), respectively. Fermentation of the pure hydrolysate by Kluyveromyces marxianus ATCC 36907 led to an ethanol yield of 61.8kg/ton(CAB), corresponding to 15 g/L ethanol and productivity of 3.75 g/( Lh). The ethanol production obtained for SSF process using K. marxianus ATCC 36907 was 18 g/L corresponding to 80% yield and 74.2kg/ton(CAB). Copyright © 2014 Elsevier Ltd. All rights reserved.

  14. Linear equations on thermal degradation products of wood chips in alkaline glycerol

    International Nuclear Information System (INIS)

    Demirbas, Ayhan

    2004-01-01

    Wood chips of 0.3 and 2 mm depth from poplar and spruce wood samples, respectively, were degraded by using glycerol as a solvent and alkaline glycerol with and without Na 2 CO 3 and NaOH catalysts at different degradation temperatures: 440, 450, 460, 470, 480, 490 and 500 K. By products from the degradation processes of the ligno celluloses include lignin degradation products. Lignin and its degradation products have fuel values. The total degradation degree and cellulose degradation of the wood chips were determined to find the relationship, if any, between the yields of total degradation degree (YTD) and degradation temperature (T). There is a good linear relationship between YTD or the yields of cellulose degradation (YCD) and T (K). For the wood samples, the regression equations from NaOH (10%) catalytic runs for 0.3 mm x 15 mm x 15 mm chip size are: For poplar wood: (YTD=0.7250T-267.507) (YCD=0.1736T-71.707) For spruce wood: (YTD=0.2650T-105.979) (YCD=0.0707T-27.507) For Eqs., the square of the correlation coefficient (r 2 ) were 0.9841, 0.9496, 0.9839 and 0.9447, respectively

  15. 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)

  16. COUPLING THE ALKALINE-SURFACTANT-POLYMER TECHNOLOGY AND THE GELATION TECHNOLOGY TO MAXIMIZE OIL PRODUCTION

    Energy Technology Data Exchange (ETDEWEB)

    Malcolm Pitts; Jie Qui; Dan Wilson; Phil Dowling

    2004-05-01

    Gelation technologies have been developed to provide more efficient vertical sweep efficiencies for flooding naturally fractured oil reservoirs or more efficient areal sweep efficiency those with high permeability contrast ''thief zones''. The field proven alkaline-surfactant-polymer technology economically recovers 15% to 25% OOIP more oil than waterflooding in the swept pore space of an oil reservoir. However, alkaline-surfactant-polymer technology is not amenable to the naturally fractured reservoirs or those with thief zones because much of the injected solution bypasses the target pore space containing oil. The objective of this work is to investigate whether combining these two technologies could broaden the applicability of alkaline-surfactant-polymer flooding into these reservoirs. Fluid-fluid interaction with different gel chemical compositions and alkaline-surfactant-polymer solution with pH values ranging from 9.2 to 12.9 have been tested. Aluminum-polyacrylamide gels are not stable to alkaline-surfactant-polymer solutions at any pH. Chromium--polyacrylamide gels with polymer to chromium ion ratios of 25 or greater were stable to alkaline-surfactant-polymer solutions if solution pH was 10.6 or less. When the polymer to chromium ion was 15 or less, chromium-polyacrylamide gels were stable to alkaline-surfactant-polymer solutions with pH values up to 12.9. Chromium-xanthan gum gels were stable to alkaline-surfactant-polymer solutions with pH values of 12.9 at the polymer to chromium ion ratios tested. Silicate-polyacrylamide, resorcinol-formaldehyde, and sulfomethylated resorcinol-formaldehyde gels were also stable to alkaline-surfactant-polymer solutions with pH values ranging from 9.2 to 12.9. Iron-polyacrylamide gels were immediately destroyed when contacted with any of the alkaline-surfactant-polymer solutions with pH values of 9.2 to 12.9.

  17. Identification, Characterization and Down-Regulation of Cysteine Protease Genes in Tobacco for Use in Recombinant Protein Production.

    Directory of Open Access Journals (Sweden)

    Kishor Duwadi

    Full Text Available Plants are an attractive host system for pharmaceutical protein production. Many therapeutic proteins have been produced and scaled up in plants at a low cost compared to the conventional microbial and animal-based systems. The main technical challenge during this process is to produce sufficient levels of recombinant proteins in plants. Low yield is generally caused by proteolytic degradation during expression and downstream processing of recombinant proteins. The yield of human therapeutic interleukin (IL-10 produced in transgenic tobacco leaves was found to be below the critical level, and may be due to degradation by tobacco proteases. Here, we identified a total of 60 putative cysteine protease genes (CysP in tobacco. Based on their predicted expression in leaf tissue, 10 candidate CysPs (CysP1-CysP10 were selected for further characterization. The effect of CysP gene silencing on IL-10 accumulation was examined in tobacco. It was found that the recombinant protein yield in tobacco could be increased by silencing CysP6. Transient expression of CysP6 silencing construct also showed an increase in IL-10 accumulation in comparison to the control. Moreover, CysP6 localizes to the endoplasmic reticulum (ER, suggesting that ER may be the site of IL-10 degradation. Overall results suggest that CysP6 is important in determining the yield of recombinant IL-10 in tobacco leaves.

  18. The mimivirus R355 gene product: preliminary crystallographic analysis of a putative ubiquitin-like protein-specific protease

    International Nuclear Information System (INIS)

    Jeudy, Sandra; Lartigue, Audrey; Mansuelle, Pascal; Ogata, Yuki; Abergel, Chantal

    2010-01-01

    The genome sequence of mimivirus, the largest known double-stranded DNA virus, encodes a putative protease: the R355 gene product. Its expression in E. coli, its crystallization and the preliminary phasing of a MAD data set using the selenium signal present in a crystal of recombinant selenomethionine-substituted protein are reported. The complete genome sequence of the largest known double-stranded DNA virus, mimivirus, reveals the presence of a gene (denoted R355) that potentially encodes a cysteine protease that is expressed late (after 6 h) in the infectious cycle of the virus. In order to verify a sequence-based functional prediction and understand its role during the infectious process, the R355 protein was produced to assay its proteolytic activity and solve its three-dimensional structure. Here, the preliminary crystallographic analysis of the recombinant viral protein is reported. The crystals belonged to the orthorhombic space group P2 1 2 1 2 1 , with a monomer in the asymmetric unit. A MAD data set was used for preliminary phasing using the selenium signal from a selenomethionine-substituted protein crystal

  19. Influence of media composition on the production of alkaline α-amylase from Bacillus subtilis CB-18.

    Science.gov (United States)

    Ogbonnaya, Nwokoro; Odiase, Anthonia

    2012-01-01

    Starch, a homopolysaccharide is an important and an abundant food reserve and energy source. Starches are processed to yield different products which find many industrial applications. Alpha-amylases hydrolyze starch by cleaving α-1,4-glucosidic bonds and have been used in food, textile and pharmaceutical industries [Sun et al. 2010]. Enzymatic conversion of starch with amylase presents an economically superior alternative to the conventional method of starch gelatinization. Alkaline α-amylase has an important position in the global enzyme market as a constituent of detergent. In this paper, we screened soil bacteria and an isolate, alkalophilic Bacillus subtilis CB-18 was found to produce an alkaline α-amylase in different media. MATERIAL AND METHODS. Screening of the isolates for amylolytic activity was carried out by growing bacteria isolated from the soil in starch agar plates and subsequently staining the plates with iodine solution to reveal zones of hydrolysis of starch. The selected isolate, Bacillus subtlis CB-18 was grown in different media at alkaline pH to evaluate the influence of media composition on alkaline α-amylase production. Enzyme assay was carried out by growing the culture in a broth medium and obtaining cell - free culture supernatant after centrifugation at 2515 × g for 15 minutes Amylase activity was determined by incubating 0.5 ml of crude enzyme solution in 0.1M Tris/HCl buffer (pH 8.5) with 0.5 ml of 1% soluble starch solution. The reaction was terminated by the addition of DNS reagent and reducing sugar produced from the amylolytic reaction was determined. Bacillus subtilis CB-18 used for this work was selected because it produced 7 mm zone diameter on starch agar plate. This organism was cultured in different alkaline broth media containing 2% soluble starch as inducer carbohydrate for α-amylase production. Among the carbon sources used for enzyme production, sorbitol was the best to stimulate enzyme production with

  20. Novel Bacillus subtilis IND19 cell factory for the simultaneous production of carboxy methyl cellulase and protease using cow dung substrate in solid-substrate fermentation.

    Science.gov (United States)

    Vijayaraghavan, Ponnuswamy; Arun, Arumugaperumal; Al-Dhabi, Naif Abdullah; Vincent, Samuel Gnana Prakash; Arasu, Mariadhas Valan; Choi, Ki Choon

    2016-01-01

    Hydrolytic enzymes, such as cellulases and proteases, have various applications, including bioethanol production, extraction of fruit and vegetable juice, detergent formulation, and leather processing. Solid-substrate fermentation has been an emerging method to utilize low-cost agricultural residues for the production of these enzymes. Although the production of carboxy methyl cellulase (CMCase) and protease in solid state fermentation (SSF) have been studied extensively, research investigating multienzyme production in a single fermentation process is limited. The production of multienzymes from a single fermentation system could reduce the overall production cost of enzymes. In order to achieve enhanced production of enzymes, the response surface methodology (RSM) was applied. Bacillus subtilis IND19 utilized cow dung substrates for the production of CMCase and protease. A central composite design and a RSM were used to determine the optimal concentrations of peptone, NaH2PO4, and medium pH. Maximum productions of CMCase and protease were observed at 0.9 % peptone, 0.78 % NaH2PO4, and medium pH of 8.41, and 1 % peptone, 0.72 % NaH2PO4, and medium pH of 8.11, respectively. Under the optimized conditions, the experimental yield of CMCase and protease reached 473.01 and 4643 U/g, which were notably close to the predicted response (485.05 and 4710 U/g). These findings corresponded to an overall increase of 2.1- and 2.5-fold in CMCase and protease productions, respectively. Utilization of cow dung for the production of enzymes is critical to producing multienzymes in a single fermentation step. Cow dung is available in large quantity throughout the year. This report is the first to describe simultaneous production of CMCase and protease using cow dung. This substrate could be directly used as the culture medium without any pretreatment for the production of these enzymes at an industrial scale.

  1. Purification and characterisation of a protease (tamarillin) from tamarillo fruit

    KAUST Repository

    Li, Zhao

    2018-02-16

    A protease from tamarillo fruit (Cyphomandra betacea Cav.) was purified by ammonium sulphate precipitation and diethylaminoethyl-Sepharose chromatography. Protease activity was determined on selected peak fractions using a casein substrate. Sodium dodecyl sulphate polyacrylamide gel electrophoresis analysis showed that the peak with the highest protease activity consisted of one protein of molecular mass ca. 70 kDa. The protease showed optimal activity at pH 11 and 60°C. It was sensitive to phenylmethylsulphonyl fluoride while ethylenediaminetetraacetic acid and p-chloromercuribenzoic acid had little effect on its activity, indicating that this enzyme was a serine protease. Hg2+ strongly inhibited enzyme activity, possibly due to formation of mercaptide bonds with the thiol groups of the protease, suggesting that some cysteine residues may be located close to the active site. De novo sequencing strongly indicated that the protease was a subtilisin-like alkaline serine protease. The protease from tamarillo has been named \\'tamarillin\\'.

  2. Purification and characterisation of a protease (tamarillin) from tamarillo fruit

    KAUST Repository

    Li, Zhao; Scott, Ken; Hemar, Yacine; Zhang, Huoming; Otter, Don

    2018-01-01

    A protease from tamarillo fruit (Cyphomandra betacea Cav.) was purified by ammonium sulphate precipitation and diethylaminoethyl-Sepharose chromatography. Protease activity was determined on selected peak fractions using a casein substrate. Sodium dodecyl sulphate polyacrylamide gel electrophoresis analysis showed that the peak with the highest protease activity consisted of one protein of molecular mass ca. 70 kDa. The protease showed optimal activity at pH 11 and 60°C. It was sensitive to phenylmethylsulphonyl fluoride while ethylenediaminetetraacetic acid and p-chloromercuribenzoic acid had little effect on its activity, indicating that this enzyme was a serine protease. Hg2+ strongly inhibited enzyme activity, possibly due to formation of mercaptide bonds with the thiol groups of the protease, suggesting that some cysteine residues may be located close to the active site. De novo sequencing strongly indicated that the protease was a subtilisin-like alkaline serine protease. The protease from tamarillo has been named 'tamarillin'.

  3. COUPLING THE ALKALINE-SURFACTANT-POLYMER TECHNOLOGY AND THE GELATION TECHNOLOGY TO MAXIMIZE OIL PRODUCTION

    Energy Technology Data Exchange (ETDEWEB)

    Malcolm Pitts; Jie Qi; Dan Wilson

    2004-10-01

    Gelation technologies have been developed to provide more efficient vertical sweep efficiencies for flooding naturally fractured oil reservoirs or more efficient areal sweep efficiency for those with high permeability contrast ''thief zones''. The field proven alkaline-surfactant-polymer technology economically recovers 15% to 25% OOIP more oil than waterflooding from swept pore space of an oil reservoir. However, alkaline-surfactant-polymer technology is not amenable to naturally fractured reservoirs or those with thief zones because much of injected solution bypasses target pore space containing oil. This work investigates whether combining these two technologies could broaden applicability of alkaline-surfactant-polymer flooding into these reservoirs. A prior fluid-fluid report discussed interaction of different gel chemical compositions and alkaline-surfactant-polymer solutions. Gel solutions under dynamic conditions of linear corefloods showed similar stability to alkaline-surfactant-polymer solutions as in the fluid-fluid analyses. Aluminum-polyacrylamide, flowing gels are not stable to alkaline-surfactant-polymer solutions of either pH 10.5 or 12.9. Chromium acetate-polyacrylamide flowing and rigid flowing gels are stable to subsequent alkaline-surfactant-polymer solution injection. Rigid flowing chromium acetate-polyacrylamide gels maintained permeability reduction better than flowing chromium acetate-polyacrylamide gels. Silicate-polyacrylamide gels are not stable with subsequent injection of either a pH 10.5 or a 12.9 alkaline-surfactant-polymer solution. Neither aluminum citrate-polyacrylamide nor silicate-polyacrylamide gel systems produced significant incremental oil in linear corefloods. Both flowing and rigid flowing chromium acetate-polyacrylamide gels produced incremental oil with the rigid flowing gel producing the greatest amount. Higher oil recovery could have been due to higher differential pressures across cores. None of

  4. Production of a Highly Protease-Resistant Fungal α-Galactosidase in Transgenic Maize Seeds for Simplified Feed Processing.

    Science.gov (United States)

    Yang, Wenxia; Zhang, Yuhong; Zhou, Xiaojin; Zhang, Wei; Xu, Xiaolu; Chen, Rumei; Meng, Qingchang; Yuan, Jianhua; Yang, Peilong; Yao, Bin

    2015-01-01

    Raffinose-family oligosaccharide (RFO) in soybeans is one of the major anti-nutritional factors for poultry and livestocks. α-Galactosidase is commonly supplemented into the animal feed to hydrolyze α-1,6-galactosidic bonds on the RFOs. To simplify the feed processing, a protease-resistant α-galactosidase encoding gene from Gibberella sp. strain F75, aga-F75, was modified by codon optimization and heterologously expressed in the embryos of transgentic maize driven by the embryo-specific promoter ZM-leg1A. The progenies were produced by backcrossing with the commercial inbred variety Zheng58. PCR, southern blot and western blot analysis confirmed the stable integration and tissue specific expression of the modified gene, aga-F75m, in seeds over four generations. The expression level of Aga-F75M reached up to 10,000 units per kilogram of maize seeds. In comparison with its counterpart produced in Pichia pastoris strain GS115, maize seed-derived Aga-F75M showed a lower temperature optimum (50 °C) and lower stability over alkaline pH range, but better thermal stability at 60 °C to 70 °C and resistance to feed pelleting inactivation (80 °C). This is the first report of producing α-galactosidase in transgenic plant. The study offers an effective and economic approach for direct utilization of α-galactosidase-producing maize without any purification or supplementation procedures in the feed processing.

  5. Inorganic nanofibers with tailored placement of nanocatalysts for hydrogen production via alkaline hydrolysis of glucose.

    Science.gov (United States)

    Hansen, Nathaniel S; Ferguson, Thomas E; Panels, Jeanne E; Park, Ah-Hyung Alissa; Joo, Yong Lak

    2011-08-12

    Monoaxial silica nanofibers containing iron species as well as coaxial nanofibers with a pure silica core and a silica shell containing high concentrations of iron nanocrystals were fabricated via electrospinning precursor solutions, followed by thermal treatment. Tetraethyl-orthosilicate (TEOS) and iron nitrate (Fe(NO(3))(3)) were used as the precursors for the silica and iron phases, respectively. Thermal treatments of as-spun precursor fibers were applied to generate nanocrystals of iron with various oxidation states (pure iron and hematite). Scanning electron microscopy (SEM), x-ray diffraction (XRD), and transmission electron microscopy (TEM) were used to probe the fiber morphology and crystal structures. The results indicated that the size, phase, and placement of iron nanocrystals can be tuned by varying the precursor concentration, thermal treatment conditions, and processing scheme. The resulting nanofiber/metal systems obtained via both monoaxial and coaxial electrospinning were applied as catalysts to the alkaline hydrolysis of glucose for the production of fuel gas. Differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), and bulk weight change in a furnace with residual gas analysis (RGA) were used to evaluate the performance of the catalysts for various ratios of both Fe to Si, and catalyst to glucose, and the oxidation state of the iron nanocrystals. The product gas is composed of mostly H(2) (>96 mol%) and CH(4) with very low concentrations of CO(2) and CO. Due to the clear separation of reaction temperature for H(2) and CH(4) production, pure hydrogen can be obtained at low reaction temperatures. Our coaxial approach demonstrates that placing the iron species selectively near the fiber surface can lead to two to three fold reduction in catalytic consumption compared to the monoaxial fibers with uniform distribution of catalysts.

  6. Biodegradation of the alkaline cellulose degradation products generated during radioactive waste disposal.

    Science.gov (United States)

    Rout, Simon P; Radford, Jessica; Laws, Andrew P; Sweeney, Francis; Elmekawy, Ahmed; Gillie, Lisa J; Humphreys, Paul N

    2014-01-01

    The anoxic, alkaline hydrolysis of cellulosic materials generates a range of cellulose degradation products (CDP) including α and β forms of isosaccharinic acid (ISA) and is expected to occur in radioactive waste disposal sites receiving intermediate level radioactive wastes. The generation of ISA's is of particular relevance to the disposal of these wastes since they are able to form complexes with radioelements such as Pu enhancing their migration. This study demonstrates that microbial communities present in near-surface anoxic sediments are able to degrade CDP including both forms of ISA via iron reduction, sulphate reduction and methanogenesis, without any prior exposure to these substrates. No significant difference (n = 6, p = 0.118) in α and β ISA degradation rates were seen under either iron reducing, sulphate reducing or methanogenic conditions, giving an overall mean degradation rate of 4.7 × 10(-2) hr(-1) (SE ± 2.9 × 10(-3)). These results suggest that a radioactive waste disposal site is likely to be colonised by organisms able to degrade CDP and associated ISA's during the construction and operational phase of the facility.

  7. Biodegradation of the alkaline cellulose degradation products generated during radioactive waste disposal.

    Directory of Open Access Journals (Sweden)

    Simon P Rout

    Full Text Available The anoxic, alkaline hydrolysis of cellulosic materials generates a range of cellulose degradation products (CDP including α and β forms of isosaccharinic acid (ISA and is expected to occur in radioactive waste disposal sites receiving intermediate level radioactive wastes. The generation of ISA's is of particular relevance to the disposal of these wastes since they are able to form complexes with radioelements such as Pu enhancing their migration. This study demonstrates that microbial communities present in near-surface anoxic sediments are able to degrade CDP including both forms of ISA via iron reduction, sulphate reduction and methanogenesis, without any prior exposure to these substrates. No significant difference (n = 6, p = 0.118 in α and β ISA degradation rates were seen under either iron reducing, sulphate reducing or methanogenic conditions, giving an overall mean degradation rate of 4.7 × 10(-2 hr(-1 (SE ± 2.9 × 10(-3. These results suggest that a radioactive waste disposal site is likely to be colonised by organisms able to degrade CDP and associated ISA's during the construction and operational phase of the facility.

  8. Production of ultrafine zinc powder from wastes containing zinc by electrowinning in alkaline solution

    Directory of Open Access Journals (Sweden)

    Zhao Youcai

    2013-12-01

    Full Text Available Production of ultrafine zinc powder from industrial wastes by electrowinning in alkaline solution was studied. Stainless steel and magnesium electrodes were used as anode and cathode, respectively. Morphology, size distribution and composition of the Zn particles were characterized by Scanning Electron Microscopy, Laser Particle Size Analyzer, and Inductive Coupled Plasma Emission Spectrometer. The required composition of the electrolyte for ultrafine particles was found to be 25-35 g/L Zn, 200-220 g/L NaOH and 20-40 mg/L Pb. The optimal conditions were a current density of 1000-1200 A/m² and an electrolyte temperature of 30-40 °C. The results indicated that the lead additive exerted a beneficial effect on the refining of the particles, by increasing the cathodic polarization. Through this study, ultrafine zinc powder with a size distribution of around 10 μm could be produced, and considerably high current efficiencies (97-99 % were obtained.

  9. Alkaline/peracetic acid as a pretreatment of lignocellulosic biomass for ethanol fuel production

    Science.gov (United States)

    Teixeira, Lincoln Cambraia

    Peracetic acid is a lignin oxidation pretreatment with low energy input by which biomass can be treated in a silo type system for improving enzymatic digestibility of lignocellulosic materials for ethanol production. Experimentally, ground hybrid poplar wood and sugar cane bagasse are placed in plastic bags and a peracetic acid solution is added to the biomass in different concentrations based on oven-dry biomass. The ratio of solution to biomass is 6:1; after initial mixing of the resulting paste, a seven-day storage period at about 20°C is used in this study. As a complementary method, a series of pre-pretreatments using stoichiometric amounts of sodium hydroxide and ammonium hydroxide based on 4-methyl-glucuronic acid and acetyl content in the biomass is been performed before addition of peracetic acid. The alkaline solutions are added to the biomass in a ratio of 14:1 solution to biomass; the slurry is mixed for 24 hours at ambient temperature. The above procedures give high xylan content substrates. Consequently, xylanase/beta-glucosidase combinations are more effective than cellulase preparations in hydrolyzing these materials. The pretreatment effectiveness is evaluated using standard enzymatic hydrolysis and simultaneous saccharification and cofermentation (SSCF) procedures. Hybrid poplar wood pretreated with 15 and 21% peracetic acid based on oven-dry weight of wood gives glucan conversion yields of 76.5 and 98.3%, respectively. Sugar cane bagasse pretreated with the same loadings gives corresponding yields of 85.9 and 93.1%. Raw wood and raw bagasse give corresponding yields of 6.8 and 28.8%, respectively. The combined 6% NaOH/15% peracetic acid pretreatments increase the glucan conversion yields from 76.5 to 100.0% for hybrid poplar wood and from 85.9 to 97.6% for sugar cane bagasse. Respective ethanol yields of 92.8 and 91.9% are obtained from 6% NaOH/15% peracetic acid pretreated materials using recombinant Zymomonas mobilis CP4/pZB5. Peracetic acid

  10. FULL ALKALINE HYDROLYSIS OF PESTICIDE DYMETOAT WITH OBTAINING ECO-SAFE PRODUCTS

    Directory of Open Access Journals (Sweden)

    А. Ранський

    2012-04-01

    Full Text Available In this work alkaline hydrolysis of the dimetoat pesticide and ways of determination of final substanceswere presented. Dependence passing of the reaction and time of the reaction was simulated

  11. Coupling the Alkaline-Surfactant-Polymer Technology and The Gelation Technology to Maximize Oil Production

    Energy Technology Data Exchange (ETDEWEB)

    Malcolm Pitts; Jie Qi; Dan Wilson; Phil Dowling; David Stewart; Bill Jones

    2005-12-01

    Gelation technologies have been developed to provide more efficient vertical sweep efficiencies for flooding naturally fractured oil reservoirs or reservoirs with different sand lenses with high permeability contrast. The field proven alkaline-surfactant-polymer technology economically recovers 15% to 25% OOIP more crude oil than waterflooding from swept pore space of an oil reservoir. However, alkaline-surfactant-polymer technology is not amenable to naturally fractured reservoirs or reservoirs with high permeability contrast zones because much of injected solution bypasses target pore space containing oil. This work investigates whether combining these two technologies could broaden applicability of alkaline-surfactant-polymer flooding into these reservoirs. Fluid-fluid interaction with different gel chemical compositions and alkaline-surfactant-polymer solution with pH values ranging from 9.2 to 12.9 have been tested. Aluminum-polyacrylamide gels are not stable to alkaline-surfactant-polymer solutions at any pH. Chromium-polyacrylamide gels with polymer to chromium ion ratios of 25 or greater were stable to alkaline-surfactant-polymer solutions if solution pH was 10.6 or less. When the polymer to chromium ion was 15 or less, chromium-polyacrylamide gels were stable to alkaline-surfactant-polymer solutions with pH values up to 12.9. Chromium-xanthan gum gels were stable to alkaline-surfactant-polymer solutions with pH values of 12.9 at the polymer to chromium ion ratios tested. Silicate-polyacrylamide, resorcinol-formaldehyde, and sulfomethylated resorcinol-formaldehyde gels were also stable to alkaline-surfactant-polymer solutions with pH values ranging from 9.2 to 12.9. Iron-polyacrylamide gels were immediately destroyed when contacted with any of the alkaline-surfactant-polymer solutions with pH values ranging from 9.2 to 12.9. Gel solutions under dynamic conditions of linear corefloods showed similar stability to alkaline-surfactant-polymer solutions as in

  12. A small-scale flow alkaline fuel cell for on-site production of hydrogen peroxide

    International Nuclear Information System (INIS)

    Brillas, Enric; Alcaide, Francisco; Cabot, Pere-Lluis

    2002-01-01

    The behavior of a small-scale flow alkaline fuel cell (AFC) built-up for on-site production of HO 2 - using commercial gas-diffusion electrodes has been studied. It produces a spontaneous current due to the oxidation of H 2 to H 2 O at the H 2 -diffusion anode and the reduction of O 2 to HO 2 - at the O 2 -diffusion cathode, while a fresh 1.0-6.0 mol dm -3 KOH electrolyte at 15.0-45.0 deg. C is injected through it. Under circulation of HO 2 - +KOH solutions in open circuit, the flow AFC behaves as a two-electron reversible system. When it is shorted with an external load (R ext ), steady cell voltage-current density curves are found. The use of O 2 /N 2 mixtures to fed the cathode causes a loss of its performance, being required to supply pure O 2 to yield a maximum HO 2 - electrogeneration. The current density and HO 2 - productivity increase with raising OH - concentration, temperature and pressure of O 2 fed. At R ext =0.10 Ω, a current efficiency close to 100% is obtained, and current densities >100 mA cm -2 are achieved for 1.0 mol dm -3 KOH at 45.0 deg. C and for higher KOH concentrations at 25.0 deg. C. The flow AFC can work under optimum conditions up to 6.0 mol dm -3 KOH and 45.0 deg. C for possible industrial applications

  13. Studies On Optimization Of Protease Production Using Bacterial Isolate Clri Strain 5468 And Its Application In Dehairing And Hydrolysis Of Tannery Fleshings Solid Waste Management

    Directory of Open Access Journals (Sweden)

    Vimala Devi Seenivasagham

    2015-08-01

    Full Text Available The strain which produces protease was originally isolated characterized in Biotechnology laboratory at CLRI and was maintained. The microorganism was growned on several proteolytic media and the maximum activity was observed. The characterization of enzyme was analysed for different pH temperature size of inoculum inhibitors age of the culture. Then the enzyme was observed for the unhairing of skin and the disadvantage in chemical treatment was studied. The conformation of unhairing was studied using histology studies. The tannery waste solid fleshings as it is cannot be directly disposed off to the environment. It was treated with the microbial proteases. The hydrolysis of waste was done using proteases. The solid waste was converted to protien fat and the salt matter. Future work is to optimize the cheap media for the production of the enzyme for large scale applications in various industries.

  14. Processing Proteases

    DEFF Research Database (Denmark)

    Ødum, Anders Sebastian Rosenkrans

    -terminal of the scissile bond, leaving C-terminal fusions to have non-native C-termini after processing. A solution yielding native C-termini would allow novel expression and purification systems for therapeutic proteins and peptides.The peptidyl-Lys metallopeptidase (LysN) of the fungus Armillaria mellea (Am) is one...... 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...

  15. A genomic survey of proteases in Aspergilli

    NARCIS (Netherlands)

    Budak, Sebnem Ozturkoglu; Zhou, M.; Brouwer, Carlo; Wiebenga, A.; Benoit, Isabelle; Di Falco, Marcos; Tsang, Adrian; de Vries, Ronald P; van den Brink, J.

    2014-01-01

    BACKGROUND: Proteases can hydrolyze peptides in aqueous environments. This property has made proteases the most important industrial enzymes by taking up about 60% of the total enzyme market. Microorganisms are the main sources for industrial protease production due to their high yield and a wide

  16. Coupling the Alkaline-Surfactant-Polymer Technology and the Gelation Technology to Maximize Oil Production

    Energy Technology Data Exchange (ETDEWEB)

    Malcolm Pitts; Jie Qi; Dan Wilson; Phil Dowling; David Stewart; Bill Jones

    2005-12-01

    Gelation technologies have been developed to provide more efficient vertical sweep efficiencies for flooding naturally fractured oil reservoirs or reservoirs with different sand lenses with high permeability contrast. The field proven alkaline-surfactant-polymer technology economically recovers 15% to 25% OOIP more crude oil than waterflooding froin swept pore space of an oil reservoir. However, alkaline-surfactant-polymer technology is not amenable to naturally fractured reservoirs or reservoirs with high permeability contrast zones because much of injected solution bypasses target pore space containing oil. This work investigates whether combining these two technologies could broaden applicability of alkaline-surfactant-polymer flooding into these reservoirs. Fluid-fluid interaction with different gel chemical compositions and alkaline-surfactant-polymer solution with pH values ranging from 9.2 to 12.9 have been tested. Aluminum-polyacrylamide gels are not stable to alkaline-surfactant-polymer solutions at any pH. Chromium-polyacrylamide gels with polymer to chromium ion ratios of 25 or greater were stable to alkaline-surfactant-polymer solutions if solution pH was 10.6 or less. When the polymer to chromium ion was 15 or less, chromium-polyacrylamide gels were stable to alkaline-surfactant-polymer solutions with pH values up to 12.9. Chromium-xanthan gum gels were stable to alkaline-surfactant-polymer solutions with pH values of 12.9 at the polymer to chromium ion ratios tested. Silicate-polyacrylamide, resorcinol-formaldehyde, and sulfomethylated resorcinol-formaldehyde gels were also stable to alkaline-surfactant-polymer solutions with pH values ranging from 9.2 to 12.9. Iron-polyacrylamide gels were immediately destroyed when contacted with any of the alkaline-surfactant-polymer solutions with pH values ranging from 9.2 to 12.9. Gel solutions under dynamic conditions of linear corefloods showed similar stability to alkaline-surfactant-polymer solutions as in

  17. Optimization of alkaline cellulase production by the marine-derived fungus Chaetomium sp. using agricultural and industrial wastes as substrates

    Digital Repository Service at National Institute of Oceanography (India)

    Ravindran, C.; Naveenan, T.; Varatharajan, G.R

    coastal waters in the form of detritus and live animals (fish, shellfish). The detritus serves as a nutrient source and is the base of an extensive primary source in the food web of the tropical marine ecosystem (Raghukumar et al. 1994). Marine fungi... for the production of alkaline cellulase enzymes using agricultural and industrial wastes such as wheat bran, rice bran, cotton seeds, sugarcane bagasse and paper as substrates. Materials and Methods Sampling of plant material and isolation of fungal...

  18. Optimizing production of hydroxyapatite from alkaline residue for removal of Pb{sup 2+} from wastewater

    Energy Technology Data Exchange (ETDEWEB)

    Yan, Yubo; Wang, YanPeng; Sun, Xiuyun, E-mail: sunxyun@njust.edu.cn; Li, Jiansheng; Shen, Jinyou; Han, Weiqing; Liu, Xiaodong; Wang, Lianjun, E-mail: wanglj@njust.edu.cn

    2014-10-30

    Highlights: • The solid waste from Soda Ash Plants was firstly converted into the high-efficiency adsorbent (O-HAP). • The response surface methodology was used to optimize the preparation conditions of O-HAP. • The O-HAP showed excellent immobilization ability for Pb{sup 2+} in both aqueous and soil medium. • The maximum adsorption capacity for Pb{sup 2+} (1429 mg/g) was considerably greater than other familiar adsorbents. - Abstract: Alkaline residue, a common solid waste generated from the ammonia-soda process for the production of soda ash, has been converted into hydroxyapatite for Pb{sup 2+} removal from wastewater. Response surface methodology was used to optimize the preparation conditions which were Ca/P (molar ratio), reaction temperature and reaction time, with the Pb{sup 2+} removal percentage as targeted response. The optimum conditions were identified to be Ca/P of 1.29, reaction temperature of 165.87 °C and reaction time of 14.5 h. Batch tests were conducted to evaluate the adsorption performance of optimum adsorbent (O-HAP), and the adsorption data were analyzed with different kinetic and isotherm models. The results showed that the pseudo-second order kinetic model and Langmuir isotherm model could best describe the adsorption of Pb{sup 2+} on O-HAP. The maximum adsorption capacity calculated from Langmuir equation was 1429 mg/g, which was greater than other familiar adsorbents. The MINTEQ results predicted that the formation of different Pb precipitates was the main mechanism in Pb{sup 2+} removal process, which was in good agreement with the kinetic and thermodynamic studies and were confirmed by the SEM-EDS and XRD analysis. In addition to aqueous medium, the O-HAP also could efficiently immobilize Pb{sup 2+} from contaminated soil.

  19. Activity of the Human Rhinovirus 3C Protease Studied in Various Buffers, Additives and Detergents Solutions for Recombinant Protein Production

    DEFF Research Database (Denmark)

    Ullah, Raheem; Shah, Majid Ali; Tufail, Soban

    2016-01-01

    stringent sequence specificity and enhanced activity. Like other proteases, activity of the human rhinovirus 3C protease can be affected in part by the buffer components and additives that are generally employed for purification and stabilization of proteins, hence, necessitate their removal by tedious...

  20. Extracellular Lipase and Protease Production from a Model Drinking Water Bacterial Community Is Functionally Robust to Absence of Individual Members.

    Directory of Open Access Journals (Sweden)

    Graham G Willsey

    Full Text Available Bacteria secrete enzymes into the extracellular space to hydrolyze macromolecules into constituents that can be imported for microbial nutrition. In bacterial communities, these enzymes and their resultant products can be modeled as community property. Our goal was to investigate the impact of individual community member absence on the resulting community production of exoenzymes (extracellular enzymes involved in lipid and protein hydrolysis. Our model community contained nine bacteria isolated from the potable water system of the International Space Station. Bacteria were grown in static conditions individually, all together, or in all combinations of eight species and exoproduct production was measured by colorimetric or fluorometric reagents to assess short chain and long chain lipases, choline-specific phospholipases C, and proteases. The exoenzyme production of each species grown alone varied widely, however, the enzyme activity levels of the mixed communities were functionally robust to absence of any single species, with the exception of phospholipase C production in one community. For phospholipase C, absence of Chryseobacterium gleum led to increased choline-specific phospholipase C production, correlated with increased growth of Burkholderia cepacia and Sphingomonas sanguinis. Because each individual species produced different enzyme activity levels in isolation, we calculated an expected activity value for each bacterial mixture using input levels or known final composition. This analysis suggested that robustness of each exoenzyme activity is not solely mediated by community composition, but possibly influenced by bacterial communication, which is known to regulate such pathways in many bacteria. We conclude that in this simplified model of a drinking water bacterial community, community structure imposes constraints on production and/or secretion of exoenzymes to generate a level appropriate to exploit a given nutrient environment.

  1. Overexpression of Aspergillus tubingensis faeA in protease-deficient Aspergillus niger enables ferulic acid production from plant material.

    Science.gov (United States)

    Zwane, Eunice N; Rose, Shaunita H; van Zyl, Willem H; Rumbold, Karl; Viljoen-Bloom, Marinda

    2014-06-01

    The production of ferulic acid esterase involved in the release of ferulic acid side groups from xylan was investigated in strains of Aspergillus tubingensis, Aspergillus carneus, Aspergillus niger and Rhizopus oryzae. The highest activity on triticale bran as sole carbon source was observed with the A. tubingensis T8.4 strain, which produced a type A ferulic acid esterase active against methyl p-coumarate, methyl ferulate and methyl sinapate. The activity of the A. tubingensis ferulic acid esterase (AtFAEA) was inhibited twofold by glucose and induced twofold in the presence of maize bran. An initial accumulation of endoglucanase was followed by the production of endoxylanase, suggesting a combined action with ferulic acid esterase on maize bran. A genomic copy of the A. tubingensis faeA gene was cloned and expressed in A. niger D15#26 under the control of the A. niger gpd promoter. The recombinant strain has reduced protease activity and does not acidify the media, therefore promoting high-level expression of recombinant enzymes. It produced 13.5 U/ml FAEA after 5 days on autoclaved maize bran as sole carbon source, which was threefold higher than for the A. tubingensis donor strain. The recombinant AtFAEA was able to extract 50 % of the available ferulic acid from non-pretreated maize bran, making this enzyme suitable for the biological production of ferulic acid from lignocellulosic plant material.

  2. Inhibition of protease activity by antisense RNA improves recombinant protein production in Nicotiana tabacum cv. Bright Yellow 2 (BY-2) suspension cells.

    Science.gov (United States)

    Mandal, Manoj K; Fischer, Rainer; Schillberg, Stefan; Schiermeyer, Andreas

    2014-08-01

    Recombinant proteins produced in plant suspension cultures are often degraded by endogenous plant proteases when secreted into the medium, resulting in low yields. To generate protease-deficient tobacco BY-2 cell lines and to retrieve the sequence information, we cloned four different protease cDNAs from tobacco BY-2 cells (NtAP, NtCP, NtMMP1, and NtSP), which represent the major catalytic classes. The simultaneous expression of antisense RNAs against these endogenous proteases led to the establishment of cell lines with reduced levels of endogenous protease expression and activity at late stages of the cultivation cycle. One of the cell lines showing reduced proteolytic activity in the culture medium was selected for the expression of the recombinant full-length IgG1(κ) antibody 2F5, recognizing the gp41 surface protein of HIV-1. This cell line showed significantly reduced degradation of the 2F5 heavy chain, resulting in four-fold higher accumulation of the intact antibody heavy chain when compared to transformed wild type cells expressing the same antibody. N-terminal sequencing data revealed that the antibody has two cleavage sites within the CDR-H3 and one site at the end of the H4-framework region. These cleavage sites are found to be vulnerable to serine proteases. The data provide a basis for further improvement of plant cells for the production of recombinant proteins in plant cell suspension cultures. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Effect of lipopolysaccharide (LPS and peptidoglycan (PGN on human mast cell numbers, cytokine production, and protease composition

    Directory of Open Access Journals (Sweden)

    Wu Yalin

    2008-08-01

    Full Text Available Abstract Background Human mast cell (HuMC maturation occurs in tissues interfacing with the external environment, exposing both mast cell progenitors and mature mast cells, to bacteria and their products. It is unknown, however, whether long- or short-term exposure to bacteria-derived toll-like receptor (TLR ligands, such as lipopolysaccharide (LPS or peptidoglycan (PGN, influences HuMC biology. Results Over 6 wks of culture, LPS had minimal effect on HuMC numbers but increased CD117, tryptase and chymase expression. PGN inhibited HuMC development. For mature mast cells, LPS in the presence of rhSCF (10 ng/ml increased CD117, tryptase, chymase and carboxypeptidase expression, primarily in CD117low HuMC. LPS decreased FcεRI expression and β-hexosaminidase release; but had no effect on LTC4 and PGD2 production. PGN reduced HuMC numbers; and CD117 and tryptase expression. IL-1β and IL-6 (in addition to IL-8 and IL-12 were detected in short-term culture supernatants of LPS treated cells, and reproduced the increases in CD117, tryptase, chymase, and carboxypeptidase expression observed in the presence of LPS. Comparative studies with mouse bone marrow-derived mast cells from wild type, but not TLR4 knockout mice, showed increases in mRNA of mouse mast cell chymases MMCP-1, MMCP-2 and MMCP-4. Conclusion PGN inhibits HuMC growth, while LPS exerts its primary effects on mature HuMC by altering cytokine production and protease composition, particularly at low concentrations of SCF. These data demonstrate the ability of bacterial products to alter HuMC mediator production, granular content, and number which may be particularly relevant at mucosal sites where HuMC are exposed to these products.

  4. Neutralization of acid mine drainage using the final product from CO2 emissions capture with alkaline paper mill waste

    International Nuclear Information System (INIS)

    Perez-Lopez, Rafael; Castillo, Julio; Quispe, Dino; Nieto, Jose Miguel

    2010-01-01

    In this study, experiments were conducted to investigate the applicability of low-cost alkaline paper mill wastes as acidity neutralizing agents for treatment of acid mine drainage (AMD). Paper wastes include a calcium mud by-product from kraft pulping, and a calcite powder from a previous study focused on sequestering CO 2 by carbonation of calcium mud. The neutralization process consisted of increase of pH by alkaline additive dissolution, decrease of metals solubility and precipitation of gypsum and poorly crystallized Fe-Al oxy-hydroxides/oxy-hydroxysulphates, which acted as a sink for trace elements to that extent that solutions reached the pre-potability requirements of water for human consumption. This improvement was supported by geochemical modelling of solutions using PHREEQC software, and observations by scanning electron microscope and X-ray diffraction of reaction products. According to PHREEQC simulations, the annual amount of alkaline additive is able to treat AMD (pH 3.63, sulphate 3800 mg L -1 , iron 348 mg L -1 ) with an average discharge of about 114 and 40 L s -1 for calcium mud and calcite powder, respectively. Likewise, given the high potential of calcium mud to sequester CO 2 and of resulting calcite powder to neutralize AMD, paper wastes could be a promising solution for facing this double environmental problem.

  5. Improving methane production from digested manure biofibers by mechanical and thermal alkaline pretreatment

    DEFF Research Database (Denmark)

    Tsapekos, Panagiotis; Kougias, Panagiotis; Frison, A.

    2016-01-01

    the effluent stream of biogas reactors. Batch and continuous experiments were conducted to evaluate the efficiency of these pretreatments. In batch experiments, the mechanical pretreatment improved the degradability up to 45%. Even higher efficiency was shown by applying thermal alkaline pretreatments...

  6. Purification and characterization of protease from Bacillus cereus ...

    African Journals Online (AJOL)

    Among them, SU12 isolate was selected due to its high enzyme production ... growth and protease production which includes different carbon and nitrogen sources, ... organism for the industrial production of the extracellular protease enzyme.

  7. Protease production during growth and autolysis of submerged Metarhizium anisopliae cultures Produção de protease durante o crescimento e análise de culturas submersas de Metarhizium anisopliae

    Directory of Open Access Journals (Sweden)

    Gilberto U.L. Braga

    1999-04-01

    Full Text Available The growth and autolysis of two strains of the entomopathogenic deuteromycete fungus Metarhizium anisopliae var. anisopliae were evaluated in medium containing casein or glucose as carbon source. Parameters such as economic coefficient and degree of autolysis were determined for each strain. Protease production was determined throughout the growth and autolysis phases of the cultures on medium under conditions of protease induction (in the presence of casein as sole source of carbon and nitrogen. The fungus was shown to utilize casein as a carbon/energy source in a more efficient manner than glucose. The autolysis shown by the strains was intense under both types of growth conditions, reaching up to 62.7% of the dry mass produced and started soon after the depletion of the exogenous carbon source. The relationship between the proteolytic activities of the two strains evaluated varied significantly (a maximum of 19.78 on the 5th day and a minimum of 2.03 on the 16th day of growth during the various growth and autolysis phases, clearly showing that the difference between the growth curves and the difference in the kinetics of enzyme production may decisively affect the process of strain selection for protease production.O crescimento e a autólise de duas linhagens do deuteromiceto entomopatogênico Metarhizium anisopliae var. anisopliae foram avaliados em meio contendo caseína ou glicose como fonte de carbono. Foram determinados parâmetros como o coeficiente econômico e o grau de autólise apresentado pelas linhagens. A produção de protease foi determinada durante todas as fases do crescimento e da autólise das culturas, em meio indutor da produção de proteases (meio contendo caseína como única fonte de carbono e de nitrogênio. Pôde-se verificar que o fungo foi capaz de utilizar a caseína como fonte de carbono/energia de maneira mais eficiente do que a glicose. A autólise apresentada pelas linhagens foi intensa em ambas as condi

  8. Alkaline peroxide pretreatment of rapeseed straw for enhancing bioethanol production by Same Vessel Saccharification and Co-Fermentation

    DEFF Research Database (Denmark)

    Karagöz, Pinar; Vaitkeviciute-Rocha, Indre; Özkan, Melek

    2012-01-01

    Alkaline peroxide pretreatment of rapeseed straw was evaluated for conversion of cellulose and hemicellulose to fermentable sugars. After pretreatment, a liquid phase called pretreatment liquid and a solid phase were separated by filtration. The neutralized pretreatment liquids were used in a co...... pretreatment combination with respect to overall ethanol production. At this condition, 5.73g ethanol was obtained from pretreatment liquid and 14.07g ethanol was produced by co-fermentation of solid fraction with P. stipitis. Optimum delignification was observed when 0.5M MgSO4 was included...... in the pretreatment mixture, and it resulted in 0.92% increase in ethanol production efficiency....

  9. Production of a novel wheat gluten hydrolysate containing dipeptidyl peptidase-IV inhibitory tripeptides using ginger protease.

    Science.gov (United States)

    Taga, Yuki; Hayashida, Osamu; Kusubata, Masashi; Ogawa-Goto, Kiyoko; Hattori, Shunji

    2017-09-01

    Wheat gluten is a Pro-rich protein complex comprising glutenins and gliadins. Previous studies have reported that oral intake of enzymatic hydrolysates of gluten has beneficial effects, such as suppression of muscle injury and improvement of hepatitis. Here, we utilized ginger protease that preferentially cleaves peptide bonds with Pro at the P 2 position to produce a novel type of wheat gluten hydrolysate. Ginger protease efficiently hydrolyzed gluten, particularly under weak acidic conditions, to peptides with an average molecular weight of ginger protease can be used as a functional food for patients with type 2 diabetes.

  10. Production and properties of an extracellular protease from thermophilic Bacillus sp Produção e propriedades de uma protease extracelular de um Bacillus sp termofílico

    Directory of Open Access Journals (Sweden)

    Wellingta Cristina Almeida do Nascimento

    2004-06-01

    Full Text Available Protease production by thermophilic Bacillus sp strain SMIA-2 cultivated in liquid cultures containing trisodium citrate reached a maximum in 9h, with levels of 1.93U/mg protein. The microorganism utilized several carbon sources for the production of protease. Starch was the best substrate, followed by trisodium citrate, citric acid and sucrose. Among the various organic and inorganic nitrogen sources, ammonium nitrate was found to be the best. Studies on the protease characterization revealed that the optimum temperature of this enzyme was 60ºC. The enzyme was stable for 2h at 30ºC, while at 40ºC and 80ºC, 14% and 84% of the original activities were lost, respectively. The optimum pH of the enzyme was found to be 8.0. After incubation of crude enzyme solution for 24h at pH 5.5, 8.0 and 9.0, a decrease of about 51%, 18% and 66% of its original activity was observed respectively. A stronger inhibitory effect was observed in the presence of K+, Hg2+and Cu2+. Hg+ resulted in the complete loss of activity at 1mM concentrations. Activity was stimulated by Mn2+ and Ca+2, indicating that these ions had a functional role in the molecular structure of the enzyme.A produção de protease pelo termofílico Bacillus sp cepa SMIA-2 cultivado em culturas líquidas contendo citrato trissódico alcançou o máximo em 9h, com níveis de 1,93U/mg de proteína. O microrganismo utilizou várias fontes de carbono para a produção da protease, sendo que o amido foi o melhor substrato seguido por citrato trissódico, ácido cítrico e sacarose. Entre as várias fontes de nitrogênio orgânico e inorgânico, o nitrato de amônio foi a melhor. Estudos sobre a caracterização da protease revelaram que a temperatura ótima desta enzima foi 60ºC. A enzima foi estável por 2h a 30ºC, enquanto a 40ºC and 80ºC, 14% e 84% da atividade original foram perdidas, respectivamente. O valor ótimo de pH encontrado para a enzima foi 8,0. Após a incubação da solu

  11. Production of Extra-Cellular Proteases from Marine Bacillus Sp. Cultured in Media Containing Ammonium Sulfate as the Sole Nitrogen Source

    Directory of Open Access Journals (Sweden)

    Seri Intan, M.

    2005-01-01

    Full Text Available Useful bacterial strains can be used to increase mineralize activity of an aquatic system. These bacteria can specifically degrade targeted compound by producing extra-cellular enzymes. Three species of Bacillus i.e. B. subtilis, B. pumilus and B. licheniformis acquired from shrimp ponds were tested for their ability to utilize ammonia and produce extracellular enzymes. These bacteria were grown in artificial seawater (30 ppt salinity and pH 7.6 supplemented with decreasing yeast extract concentration but increasing ammonium sulfate concentration. All three bacteria grew in artificial seawater containing only 0.01% yeast extract and 1% ammonium sulfate. However, only B. pumilus and B. licheniformis were able to grow in the medium containing only 1% ammonium sulfate as a sole energy source. Bacterialgrowth reduced when alkaline proteases activities was maximum from culture filtrates of all three bacterial cultures during 24 hour culturing in artificial seawater containing 0.01% yeast extract and 1% ammonium sulfate at 30 C when assayed at pH 9. Bacterial growth increased when acid proteases activities was maximum from culture filtrates of all three bacterial cultures during 48 hour culturing in artificial seawater containing 0.01% yeast extract and 1% ammoniumsulfate at 30 C when assayed at pH 5.

  12. Dynamics of Phylogenetic Diversity and Its Influence on the Production of Extracellular Protease by Moderately Halotolerant Alkaliphilic Bacteria Acinetobacter Baumannii GTCR407 Nov

    Directory of Open Access Journals (Sweden)

    Thiyagarajan Gurunathan

    2010-07-01

    Full Text Available New characters emerge in the population of microorganisms living in the extreme environments due to its adaptation to ecological association. The microorganisms living in saline habitat utilize complex nutrients by adopting different strategies in Deoxyribonucleic Acid (DNA and Ribonucleic Acid (RNA, which are related to their metabolic and ecological diversities. Isolation and characterization of the organisms producing extracellular protease from such environment were the prime focus of this investigation, which can indicate the importance of metabolic diversity in phylogeny. Norberg medium was used to isolate halotolerant microorganisms from salt-cured skin. The isolates were screened for high activity of protease and the strain showing maximum activity of protease was taken for further studies. The biochemical characterization and 16s ribosomal RNA sequencing studies confirm that the isolate is Acinetobacter baumannii. Moreover, hydrolysis positive for starch and casein, negative for gelatin shows that the organism is a variant form of A. baumannii. Cell growth parameters such as pH and temperature were optimized and their values are 8 and 37oC respectively. The extracellular production of protease was optimized in the suitable medium and its enzyme activity was 165μg/ml/min. The results imply that the isolate had acquired operational genes through lateral gene transfer (LGT probably from unrelated species in the environment. This indicates that the isolate identified possesses metabolic and ecological diversities with values of phylogenetic delineation

  13. Transport phenomena in alkaline direct ethanol fuel cells for sustainable energy production

    Science.gov (United States)

    An, L.; Zhao, T. S.

    2017-02-01

    Alkaline direct ethanol fuel cells (DEFC), which convert the chemical energy stored in ethanol directly into electricity, are one of the most promising energy-conversion devices for portable, mobile and stationary power applications, primarily because this type of fuel cell runs on a carbon-neutral, sustainable fuel and the electrocatalytic and membrane materials that constitute the cell are relatively inexpensive. As a result, the alkaline DEFC technology has undergone a rapid progress over the last decade. This article provides a comprehensive review of transport phenomena of various species in this fuel cell system. The past investigations into how the design and structural parameters of membrane electrode assemblies and the operating parameters affect the fuel cell performance are discussed. In addition, future perspectives and challenges with regard to transport phenomena in this fuel cell system are also highlighted.

  14. Volatile Fatty Acids Production from Codigestion of Food Waste and Sewage Sludge Based on β-Cyclodextrins and Alkaline Treatments.

    Science.gov (United States)

    Yang, Xue; Liu, Xiang; Chen, Si; Liu, Guangmin; Wu, Shuyan; Wan, Chunli

    2016-01-01

    Volatile fatty acids (VFAs) are preferred valuable resources, which can be produced from anaerobic digestion process. This study presents a novel technology using β -cyclodextrins ( β -CD) pretreatment integrated alkaline method to enhance VFAs production from codigestion of food waste and sewage sludge. Experiment results showed that optimized ratio of food waste to sewage sludge was 3 : 2 because it provided adequate organic substance and seed microorganisms. Based on this optimized ratio, the integrated treatment of alkaline pH 10 and β -CD addition (0.2 g/g TS) performed the best enhancement on VFAs production, and the maximum VFAs production was 8631.7 mg/L which was 6.13, 1.38, and 1.57 times higher than that of control, initial pH 10, and 0.2 g β -CD/g TS treatment, respectively. Furthermore, the hydrolysis rate of protein and polysaccharides was greatly improved in integration treatment, which was 1.18-3.45 times higher than that of other tests. Though the VFAs production and hydrolysis of polymeric organics were highly enhanced, the primary bacterial communities with different treatments did not show substantial differences.

  15. Production of fibrinolytic protease from Streptomyces lusitanus isolated from marine sediments

    Science.gov (United States)

    SudeshWarma, S.; Merlyn keziah, S.; Subathra Devi, C.

    2017-11-01

    This study aim was to isolate, screen, characterize and optimize marine Streptomyces for fibrinolytic enzyme production. The potent actinomycete isolate was subjected to optimization. The parameters for optimization included pH, temperature, carbon, nitrogen sources. The crude supernatant produced was purified using size exclusion gel filtration chromatography. The optimized parameters for maximum productivity were found to be pH 7, 37°C, maltose and peptone respectively. The molecular weight of the purified enzyme was found to be 21kDa.

  16. Role of ethanol on growth, laccase production and protease activity in Pycnoporus cinnabarinus ss3

    OpenAIRE

    Meza, Juan Carlos; Auria, Richard; Lomascolo, A.; Sigoillot, J. C.; Casalot, Laurence

    2007-01-01

    Laccase production by the strain Pycnoporus cinnabarinus ss3 was studied in a solid-state culture on sugar-cane bagasse using chemical compounds as inducers (ethanol, methanol, veratryl alcohol and ferulic acid). Laccase productions were about 5- to 8.5-fold higher than non-induced cultures. Liquid-culture experiments with "Glabeled ethanol were conducted. Ninety-eight percent of the initial amount of C-14 from ethanol was recovered as (CO2)-C-14, C-14-biomass and soluble C-14-compounds (main...

  17. Orogenic potassic mafic magmatism, a product of alkaline-peraluminous mixing ? Variscan 'calc-alkaline' rocks from the Central Iberian and Ossa Morena Zones, Central Spain.

    Science.gov (United States)

    Scarrow, Jane H.; Cambeses, Aitor; Bea, Fernando; Montero, Pilar; Molina, José F.; Moreno, Juan Antonio

    2013-04-01

    Orogenic magmatic rocks provide information about mantle and crust melt-generation and -interaction processes. In this context, minor potassic mafic stocks which are formed of enriched mantle and crustal components and are common as late-orogenic intrusions in granitic plutons give insight into the timing of new crust formation and crustal recycling. Potassic mafic stocks are prevalent, albeit low volume, constituents of granite batholiths all through the European Variscan (350-280 Ma). In the Central Iberia Zone, Spanish Central System, crustal-melt, S-type, granitoid plutons are intruded by minor concomitant ultramafic-intermediate appinitic-vaugneritic stocks. Notwithstanding their whole-rock calc-alkaline composition, the stocks apparently did not have a subduction-related origin. Recent studies have attributed their genesis to mixing of alkaline mantle and peraluminous crustal melts. Their primary alkaline character, as indicated by amphibole and biotite mineral chemistry data, points, rather, towards an extension-related genesis. In the Ossa Morena Zone, south of the Central Iberian Zone, the igneous rocks also have a whole-rock calc-alkaline composition which has been considered to be the result of northward subduction of the South Portuguese Zone. Nevertheless, identification of a 'sill' of significant volume of mafic magma in the middle crust, the ´IBERSEIS reflective body', in a seismic profile across the Ossa Morena and South Portuguese Zones has cast doubt upon the calc-alkaline magmatism-subduction model; leading, instead, to the magmatism being attributed to intra-orogenic extension related to a mantle plume active from 340 Ma to 330 Ma. The aim here, then, is to reinvestigate the petrogenesis and age of the calc-alkaline rocks of the Ossa Morena Zone to determine their tectonomagmatic context be it subduction-, plume- or extension-related, and establish what they may reveal about mantle-crust interactions. Focussing, initially, on the Valencia del

  18. In-situ Alkaline Transesterification of Jatropha Curcas Seed Oil for Production of Biodiesel and Nontoxic Jatropha Seed Cake

    OpenAIRE

    Nazir, Novizar; Mangunwidjaja, Djumali; Setyaningsih, Dwi; Yuliani, Sri; Yarmo, Mohd. Ambar; Salimon, Jumat; Ramli, Nazaruddin

    2014-01-01

    The production of fatty acid methyl ester (FAME) by direct in situ alkaline-catalyzed transesterification of the triglycerides (TG) in Jatropha curcas seeds was examined. The experimental results showed that the amount of Jatropha curcas seed oil dissolved in methanol was approximately 83% of the total oil and the conversion of this oil could achieve 98% under the following conditions: less than 2% moisture content in Jatropha curcas seed flours, 0.3–0.335 mm particle size, 0.08 mol/L NaOH co...

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

    Directory of Open Access Journals (Sweden)

    Nadia Andrea Andreani

    2016-12-01

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

  20. Production of lipase and protease from an indigenous Pseudomonas aeruginosa strain and their evaluation as detergent additives: compatibility study with detergent ingredients and washing performance.

    Science.gov (United States)

    Grbavčić, Sanja; Bezbradica, Dejan; Izrael-Živković, Lidija; Avramović, Nataša; Milosavić, Nenad; Karadžić, Ivanka; Knežević-Jugović, Zorica

    2011-12-01

    An indigenous Pseudomonas aeruginosa strain has been studied for lipase and protease activities for their potential application in detergents. Produced enzymes were investigated in order to assess their compatibility with several surfactants, oxidizing agents and commercial detergents. The crude lipase appeared to retain high activity and stability in the presence of several surfactants and oxidizing agents and it was insusceptible to proteolysis. Lutensol® XP80 and Triton® X-100 strongly activated the lipase for a long period (up to 40 and 30% against the control after 1h) while the protease activity was enhanced by the addition of Triton® WR1339 and Tween® 80. The washing performance of the investigated surfactants was significantly improved with the addition of the crude enzyme preparation. Studies were further undertaken to improve enzymes production. The optimization of fermentation conditions led to an 8-fold increase of lipase production, while the production of protease was enhanced by 60%. Copyright © 2011 Elsevier Ltd. All rights reserved.

  1. Concomitant production of detergent compatible enzymes by Bacillus flexus XJU-1.

    Science.gov (United States)

    Niyonzima, Francois N; More, Sunil S

    2014-01-01

    A soil screened Bacillus flexus XJU-1 was induced to simultaneously produce alkaline amylase, alkaline lipase and alkaline protease at their optimum levels on a common medium under submerged fermentation. The basal cultivation medium consisted of 0.5% casein, 0.5% starch and 0.5% cottonseed oil as an inducer for protease, amylase, and lipase, respectively. The casein also served as nitrogen source for all 3 enzymes. The starch was also found to act as carbon source additive for both lipase and protease. Maximum enzyme production occurred on fermentation medium with 1.5% casein, 1.5% soluble starch, 2% cottonseed oil, 2% inoculum size, initial pH of 11.0, incubation temperature of 37 °C and 1% soybean meal as a nitrogen source supplement. The analysis of time course study showed that 24 h was optimum incubation time for amylase whereas 48 h was the best time for both lipase and protease. After optimization, a 3.36-, 18.64-, and 27.33-fold increase in protease, amylase and lipase, respectively was recorded. The lipase was produced in higher amounts (37.72 U/mL) than amylase and protease about 1.27 and 5.85 times, respectively. As the 3 enzymes are used in detergent formulations, the bacterium can be commercially exploited to secrete the alkaline enzymes for use in detergent industry. This is the first report for concomitant production of 3 alkaline enzymes by a bacterium.

  2. Insecticide resistance and intracellular proteases.

    Science.gov (United States)

    Wilkins, Richard M

    2017-12-01

    Pesticide resistance is an example of evolution in action with mechanisms of resistance arising from mutations or increased expression of intrinsic genes. Intracellular proteases have a key role in maintaining healthy cells and in responding to stressors such as pesticides. Insecticide-resistant insects have constitutively elevated intracellular protease activity compared to corresponding susceptible strains. This increase was shown for some cases originally through biochemical enzyme studies and subsequently putatively by transcriptomics and proteomics methods. Upregulation and expression of proteases have been characterised in resistant strains of some insect species, including mosquitoes. This increase in proteolysis results in more degradation products (amino acids) of intracellular proteins. These may be utilised in the resistant strain to better protect the cell from stress. There are changes in insect intracellular proteases shortly after insecticide exposure, suggesting a role in stress response. The use of protease and proteasome inhibitors or peptide mimetics as synergists with improved application techniques and through protease gene knockdown using RNA interference (possibly expressed in crop plants) may be potential pest management strategies, in situations where elevated intracellular proteases are relevant. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

  3. Evaluation of alkaline dissolution of Al 6061 and Al 1050 for the production of Mo-99 from LEU targets

    International Nuclear Information System (INIS)

    Mindrisz, Ana C.; Camilo, Ruth L.; Araujo, Izilda C.; Forbicini, Christina A.L.G. de O.

    2013-01-01

    Since 2008, due to the global crisis in the production of radioisotope 99 Mo, which product of decay, 99m Tc, is the tracer element most often used in nuclear medicine and accounts for about 80% of all diagnostic procedures in vivo. Studies on the alkaline dissolution to obtain 9 9M o from irradiated UAl x -Al LEU targets are under development. Processing time should be minimized, considering the short half-life of 99 Mo and 99m Tc, about 66 h and 6 h, respectively. This makes dissolution time a significant factor in the development of the process. This paper presents the results of alkaline dissolution of 'scraps' of Al 6061 and 1050, used to simulate the dissolution process of UAl x -Al targets. Dissolution time and gas releasing were evaluated using the following alkaline solutions: a) NaOH 3 mol.L -1 and NaNO 3 2 mol.L -1 , b) NaOH 3 mol.L -1 and NaNO 3 4 mol.L -1 . The initial temperature of dissolution was 85 deg C in all cases. Al 6061 showed values of dissolution time greater than that for Al 1050, 25% for NaNO 3 2 mol.L -1 and 104.55% for NaNO 3 4 mol.L -1 . The dissolution with NaNO 3 2 mol.L -1 showed that the gas releasing for Al 6061 was 2.7% greater than for Al 1050. However Al 1050 showed that gas releasing 9.92% greater than for Al 6061 during the dissolution with NaNO 3 4 mol.L -1 . The decision about what type of alloy has to be used, Al 1050 or Al 6061, it will be upto the group that will manufacture the targets for the RMB. (author)

  4. Biodegradation of a keratin waste and the concomitant production of detergent stable serine proteases from Paecilomyces lilacinus.

    Science.gov (United States)

    Cavello, I A; Cavalitto, S F; Hours, R A

    2012-07-01

    Paecilomyces lilacinus (LPS 876) efficiently degraded keratin in chicken feather during submerged cultivation producing extracellular proteases. Characterization of crude protease activity was done including its compatibility in commercial detergents. Optimum pH and temperature were 10.0 and 60 °C, respectively. Protease activity was enhanced by Ca²⁺ but was strongly inhibited by PMSF and by Hg²⁺ suggesting the presence of thiol-dependent serine proteases. The crude protease showed extreme stability toward non-ionic (Tween 20, Tween 85, and Triton X-100) and anionic (SDS) surfactants, and relative stability toward oxidizing agent (H₂O₂ and sodium perborate). In addition, it showed excellent stability and compatibility with various solid and liquid commercial detergents from 30 to 50 °C. The enzyme preparation retained more than 95% of its initial activity with solid detergents (Ariel™ and Drive™) and 97% of its original activity with a liquid detergent (Ace™) after pre-incubation at 40 °C. The protective effect of polyols (propylene glycol, PEG 4000, and glycerol) on the heat inactivation was also examined and the best results were obtained with glycerol from 50 to 60 °C. Considering its promising properties, P. lilacinus enzymatic preparation may be considered as a candidate for use in biotechnological processes (i.e., as detergent additive) and in the processing of keratinous wastes.

  5. Determination of phosphate compounds in meat products by 31-Phosphorus Nuclear Magnetic Resonance spectroscopy with methylenediphosphonic acid after alkaline extraction

    International Nuclear Information System (INIS)

    Hrynczyszyn, P.; Jastrzebska, A.; Szlyk, E.

    2010-01-01

    Modification of the extraction procedure and application of the 31 P NMR method for the determination of polyphosphates in meat products were studied. In the elaborated procedure threefold water extraction at alkaline pH (borate buffer and 0.1 M EDTA) was applied. Furthermore, the new external standard for 31 P NMR determination of phosphates was proposed. Obtained recoveries were between 95 and 99% and variation coefficients (CV) was ≤5%, indicating an increase in accuracy and the precision of the proposed procedure in relation to the spectrophotometric method. The described procedure of sample preparation with 31 P NMR method was applied for the determination of polyphosphate additives in meat products. The satisfactory precision (CV = 0.39-3.40%) shows the benefit of the NMR method in the routine analysis of the phosphate ions in meat products.

  6. Fast and efficient nanoshear hybrid alkaline pretreatment of corn stover for biofuel and materials production

    International Nuclear Information System (INIS)

    Wang, Wei; Ji, Shaowen; Lee, Ilsoon

    2013-01-01

    We report a fast and efficient nano-scale shear hybrid alkaline (NSHA) pretreatment method of lignocellulosic biomass. In this work, corn stover was pretreated in a modified Taylor–Couette reactor with alkali (sodium hydroxide) at room temperature for two minutes. Up to 82% of high cellulose content in the remaining solids was achieved with the novel NSHA pretreatment process. Compared with untreated corn stover, an approximately 4-fold increase in enzymatic cellulose conversion and a 5-fold increase in hemicellulose conversion were achieved. Compositional analysis proved significant removals of both lignin and hemicellulose after the NSHA pretreatment. SEM images revealed that the synergistic effect of NSHA pretreatment caused the severe disruption of biomass structure and exposure of cellulose microfibril aggregates in NSHA pretreated corn stover. Highlights: ► A fast nanoshear hybrid alkaline (NSHA) pretreatment method is reported. ► A modified Taylor–Couette reactor was applied. ► The retention time of the NSHA method is only 2 min. ► A 100% conversion of glucan was achieved in one day. ► NSHA greatly removed both lignin and xylan

  7. Use of alkaline flyash-based products to amend acid soils: Plant growth response and nutrient uptake

    Energy Technology Data Exchange (ETDEWEB)

    Spark, K.M.; Swift, R.S. [University of Queensland, Gatton, Qld. (Australia)

    2008-07-01

    Vast quantities of flyash are generated annually by the burning of coal in the power industry, with most of this material being stockpiled with little prospect of being utilised at present. Two alkaline flyash-based products (FAP) for use as soil amendments (FAP1 and FAP2) have been assessed using glasshouse pot trials to determine the suitability of using these products to treat acid soils. The products both contain about 80% flyash which originated from coal-fired electricity generation. The acid soils used in the study were 2 Podsols and a Ferrosol, all originating from south-east Queensland and ranging in pH (1 : 5 suspension in water) from 4 to 5.5. The flyash products when applied to the soil significantly enhanced growth of maize plants (Zea mays L.), with optimal application rates in the range 1.25-5% w/w. The FAP/soil mixtures and plants were analysed using a range of methods including extraction with DTPA, and plant biomass (aboveground dry matter). The results indicate that in addition to the liming effect, the flyash in the alkaline flyash products may enhance plant growth as a result of increasing the uptake of micro-nutrients such as copper, zinc, and manganese. The study suggests that flyash has the potential to be used as a base material in the production of soil amendment materials that can change soil pH and act as a fertiliser for certain soil micro-nutrients such as Cu, Mn, and Zn.

  8. Optimization of Amylase and Protease Production from Aspergillus awamori in Single Bioreactor Through EVOP Factorial Design Technique

    Directory of Open Access Journals (Sweden)

    Sangeeta Negi

    2006-01-01

    Full Text Available Evolutionary operation (EVOP factorial design technique was explored in order to economically produce amylase and protease at their optimum level in a single bioreactor by modified solid-state fermentation. Maximum yields of amylase and protease were achieved, using wheat bran as a substrate by a highly potent, locally isolated strain of Aspergillus awamori: Nakazawa MTCC 6652. The strain had been induced previously, inferring the ability to produce both enzymes concomitantly in a single bioreactor with their maximum capacity. The highest secretion of amylase and protease were measured to be 9420.6 and 1930 U/g, respectively, at 37 °C. pH and relative humidity were found to be optimum at 4 and 85 %, evaluated through EVOP method.

  9. Production of zinc and manganese oxide particles by pyrolysis of alkaline and Zn-C battery waste.

    Science.gov (United States)

    Ebin, Burçak; Petranikova, Martina; Steenari, Britt-Marie; Ekberg, Christian

    2016-05-01

    Production of zinc and manganese oxide particles from alkaline and zinc-carbon battery black mass was studied by a pyrolysis process at 850-950°C with various residence times under 1L/minN2(g) flow rate conditions without using any additive. The particular and chemical properties of the battery waste were characterized to investigate the possible reactions and effects on the properties of the reaction products. The thermodynamics of the pyrolysis process were studied using the HSC Chemistry 5.11 software. The carbothermic reduction reaction of battery black mass takes place and makes it possible to produce fine zinc particles by a rapid condensation, after the evaporation of zinc from a pyrolysis batch. The amount of zinc that can be separated from the black mass is increased by both pyrolysis temperature and residence time. Zinc recovery of 97% was achieved at 950°C and 1h residence time using the proposed alkaline battery recycling process. The pyrolysis residue is mainly MnO powder with a low amount of zinc, iron and potassium impurities and has an average particle size of 2.9μm. The obtained zinc particles have an average particle size of about 860nm and consist of hexagonal crystals around 110nm in size. The morphology of the zinc particles changes from a hexagonal shape to s spherical morphology by elevating the pyrolysis temperature. Copyright © 2015 Elsevier Ltd. All rights reserved.

  10. Granulocyte-macrophage colony-stimulating factor primes interleukin-13 production by macrophages via protease-activated receptor-2.

    Science.gov (United States)

    Aoki, Manabu; Yamaguchi, Rui; Yamamoto, Takatoshi; Ishimaru, Yasuji; Ono, Tomomichi; Sakamoto, Arisa; Narahara, Shinji; Sugiuchi, Hiroyuki; Hirose, Eiji; Yamaguchi, Yasuo

    2015-04-01

    Chronic inflammation is often linked to the presence of type 2-polarized macrophages, which are induced by the T helper type 2 cytokines interleukin-4 and interleukin-13 (IL-13). IL-13 is a key mediator of tissue fibrosis caused by T helper type 2-based inflammation. Human neutrophil elastase (HNE) plays a pivotal role in the pathogenesis of pulmonary fibrosis. This study investigated the priming effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) on IL-13 expression by macrophages stimulated with HNE. Adherent macrophages were obtained from primary cultures of human mononuclear cells. Expression of IL-13 mRNA and protein by GM-CSF-dependent macrophages was investigated after stimulation with HNE, using the polymerase chain reaction and enzyme-linked immunosorbent assay. GM-CSF had a priming effect on IL-13 mRNA and protein expression by macrophages stimulated with HNE, while this effect was not observed for various other cytokines. GM-CSF-dependent macrophages showed a significant increase in the expression of protease activated receptor-2 (PAR-2) mRNA and protein. The response of IL-13 mRNA to HNE was significantly decreased by pretreatment with alpha1-antitrypsin, a PAR-2 antibody (SAM11), or a PAR-2 antagonist (ENMD-1068). These findings suggest that stimulation with HNE can induce IL-13 production by macrophages, especially GM-CSF-dependent macrophages. Accordingly, neutrophil elastase may have a key role in fibrosis associated with chronic inflammation. Copyright © 2015 Elsevier Inc. All rights reserved.

  11. Alkaline conditions stimulate the production of 1,3-propanediol in Lactobacillus panis PM1 through shifting metabolic pathways.

    Science.gov (United States)

    Grahame, Douglas A S; Kang, Tae Sun; Khan, Nurul H; Tanaka, Takuji

    2013-07-01

    A novel Lactobacillus panis PM1 isolate was found to be capable of converting glycerol to 1,3-propanediol (1,3-PDO), an increasingly valuable commodity chemical. In this study the effects of various process parameters, including glucose and glycerol concentrations, inoculum size, temperature, aeration, pH, and carbon source were examined to determine the optimal conditions for the production of 1,3-PDO using a culture method simulating late log to early stationary phases. Inoculum size did not influence the production of 1,3-PDO, and temperature variance showed similar 1,3-PDO production between 25 and 37 °C under the examined conditions. Glycerol concentration and pH played a primary role in the final concentration of 1,3-PDO. The highest production occurred at 150-250 mM glycerol when 50 mM glucose was available. Alkaline initial conditions (pH 9-10) stimulated the production of 1,3-PDO which concurrently occurred with increased acetic acid production. Under these conditions, 213.6 mM of 1,3-PDO were produced from 300 mM glycerol (conversion efficiency was 71 %). These observations indicated that the production of 1,3-PDO was associated with the shift of the metabolic end-product ethanol to acetic acid, and that this shift resulted in an excess concentration of NADH available for the processing of glycerol to 1,3-PDO.

  12. 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

    2013-12-01

    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.

  13. Optimization of aqueous enzymatic extraction of oil from shrimp processing by-products using response surface methodology

    Directory of Open Access Journals (Sweden)

    Chen WENWEI

    2018-06-01

    Full Text Available Abstract The aqueous enzymatic extraction (AEE of oil from shrimp processing by-products was investigated. Four kinds of proteases, including alkaline protease, neutral protease, flavor protease and compound protease, were applied to hydrolysis shrimp processing by-products. The results showed that flavor protease was the best hydrolysis enzyme for shrimp processing by-products to obtain high oil recovery. The influences of four factors, including enzyme amount, liquid/solid ratio, hydrolysis time and hydrolysis temperature, on shrimp oil extraction yield were also studied. The flavor enzyme hydrolysis condition was optimized as following: enzyme amount of 2.0% (w/w, liquid/solid ratio of 9.0ml/g, hydrolysis time of 2.6 h and hydrolysis temperature of 50 °C. Under these optimum hydrolysis conditions, the experimental oil extraction yield was 88.9%.

  14. Isolation of a novel alkaline-stable lipase from a metagenomic library and its specific application for milkfat flavor production.

    Science.gov (United States)

    Peng, Qing; Wang, Xu; Shang, Meng; Huang, Jinjin; Guan, Guohua; Li, Ying; Shi, Bo

    2014-01-04

    Lipolytic enzymes are commonly used to produce desired flavors in lipolyzed milkfat (LMF) manufacturing processes. However, the choice of enzyme is critical because it determines the final profile of fatty acids released and the consequent flavor of the product. We previously constructed a metagenomic library from marine sediments, to explore the novel enzymes which have unique properties useful in flavor-enhancing LMF. A novel lipase Est_p6 was isolated from a metagenomic library and was expressed highly in E.coli. Bioinformatic analysis indicated that Est_p6 belongs to lipolytic enzyme family IV, the molecular weight of purified Est_p6 was estimated at 36 kDa by SDS-PAGE. The hydrolytic activity of the enzyme was stable under alkaline condition and the optimal temperature was 50°C. It had a high specific activity (2500 U/mg) toward pNP butyrate (pNP-C4), with K(m) and V(max) values of 1.148 mM and 3497 μmol∙min⁻¹∙mg⁻¹, respectively. The enzyme activity was enhanced by DTT and was not significantly inhibited by PMSF, EDTA or SDS. This enzyme also showed high hydrolysis specificity for myristate (C14) and palmitate (C16). It seems that Est_p6 has safety for commercial LMF flavor production and food manufacturing processes. The ocean is a vast and largely unexplored resource for enzymes. According the outstanding alkaline-stability of Est_p6 and it produced myristic acid and palmitic acid more efficiently than other free fatty acids in lipolyzed milkfat. This novel lipase may be used to impart a distinctive and desirable flavor and odor in milkfat flavor production.

  15. Production of Steel Casts in Two-Layer Moulds with Alkaline Binders Part 2. Facing sand with the alkaline organic binder REZOLIT

    Directory of Open Access Journals (Sweden)

    M. Holtzer

    2011-04-01

    Full Text Available This paper constitutes the second part of the article concerning the implementation of the two-layer mould technology for steel casts inZ.M. POMET. The results of the laboratory examinations of the backing sand with the inorganic binder RUDAL were presented in thefirst part of the paper. Whereas in the second part the results of the laboratory testing of the facing sand with the alkaline resin REZOLITare given. The technology of two-layer moulds was already implemented in Z.M. POMET within the target project. Examples of castingsmade in this technology are shown in the final part of this paper.

  16. Optimizing protease production from an isolate of the nematophagous fungus Duddingtonia flagrans using response surface methodology and its larvicidal activity on horse cyathostomins.

    Science.gov (United States)

    Braga, F R; Araújo, J V; Soares, F E F; Araujo, J M; Genier, H L A; Silva, A R; Carvalho, R O; Queiroz, J H; Ferreira, S R

    2011-06-01

    Protease production from Duddingtonia flagrans (isolate AC001) was optimized and the larvicidal activity of the enzymatic extract was evaluated on infective horse cyathostomin larvae (L3). Duddingtonia flagrans was grown in liquid medium with eight different variables: glucose, casein, bibasic potassium phosphate (K2HPO4), magnesium sulphate (MgSO4), zinc sulphate (ZnSO4), ferrous sulphate (FeSO4), copper sulphate (CuSO4) and temperature. The Plackett-Burman analysis showed a significant influence of MgSO4, CuSO4 and casein (P < 0.05) on protease production by D. flagrans in liquid medium. Central composite design indicated that the highest proteolytic activity was 39.56 U/ml as a function of the concentrations of casein (18.409 g/l), MgSO4 (0.10 g/l) and CuSO4 (0.50 mg/l). A significant difference (P < 0.01) was found for the larval number between the treated and control groups at the end of the experiment. A reduction of 95.46% in the number of free-living larvae was found in the treated group compared with the control. The results of this study suggest that protease production by D. flagrans (AC001) in liquid medium was optimized by MgSO4, CuSO4 and casein, showing that the optimized enzymatic extract exerted larvicidal activity on cyathostomins and therefore may contribute to large-scale industrial production.

  17. Improving methane production from anaerobic digestion of Pennisetum Hybrid by alkaline pretreatment.

    Science.gov (United States)

    Kang, Xihui; Sun, Yongming; Li, Lianhua; Kong, Xiaoying; Yuan, Zhenhong

    2018-05-01

    Alkaline pretreatment with NaOH was used to improve methane yield from Pennisetum Hybrid. The pretreatments were carried out with different NaOH solutions (2-8% w/w) at three temperatures (35, 55 and 121 °C) for different periods of time (24, 24 and 1 h). All treated and untreated Pennisetum Hybrid were digested under mesophilic conditions (37 °C) to biogas, significant effects of the pretreatments on the yield of methane were observed. Results showed the modified Gompertz equation was reliable (determination coefficients (R 2 ) greater than 0.96) to describe the kinetic behavior of anaerobic digestion of Pennisetum Hybrid. The best result, obtained by the treatment at 35 °C 2% NaOH for 24 h, resulted in the methane yield of 301.7 mL/g VS, corresponding to 21.0% improvement in the methane yield. Compositional, SEM, XRD and FTIR analysis confirmed that lignin removal, structural modification and cellulose crystalline variation were responsible for the improvement. Copyright © 2018 Elsevier Ltd. All rights reserved.

  18. Production of Steel Casts in Two-Layer Moulds with Alkaline Binders Part 1. Backing sand with the alkaline inorganic binder RUDAL

    Directory of Open Access Journals (Sweden)

    M. Holtzer

    2011-04-01

    Full Text Available Steel casts in Z.N. POMET were produced in moulds made of the moulding sand Floster. This sand did not have good knocking outproperties, required a significant binder addition (4.5-5.0 parts by weight, and the casting surface quality gave rise to clients objections.Therefore a decision of implementing two-layer moulds, in which the facing sand would consist of the moulding sand with an alkalineorganic binder while the backing sand would be made of the moulding sand with an inorganic binder also of an alkaline character - wasundertaken. The fraction of this last binder in the moulding sand mass would be smaller than that of the binder used up to now (waterglass. The application of two moulding sands of the same chemical character (highly alkaline should facilitate the reclamation processand improve the obtained reclaimed material quality, due to which it would be possible to increase the reclaim fraction in the mouldingsand (up to now it was 50%. The results of the laboratory investigations of sands with the RUDAL binder are presented in the paper.

  19. Advances in protease engineering for laundry detergents.

    Science.gov (United States)

    Vojcic, Ljubica; Pitzler, Christian; Körfer, Georgette; Jakob, Felix; Ronny Martinez; Maurer, Karl-Heinz; Schwaneberg, Ulrich

    2015-12-25

    Proteases are essential ingredients in modern laundry detergents. Over the past 30 years, subtilisin proteases employed in the laundry detergent industry have been engineered by directed evolution and rational design to tailor their properties towards industrial demands. This comprehensive review discusses recent success stories in subtilisin protease engineering. Advances in protease engineering for laundry detergents comprise simultaneous improvement of thermal resistance and activity at low temperatures, a rational strategy to modulate pH profiles, and a general hypothesis for how to increase promiscuous activity towards the production of peroxycarboxylic acids as mild bleaching agents. The three protease engineering campaigns presented provide in-depth analysis of protease properties and have identified principles that can be applied to improve or generate enzyme variants for industrial applications beyond laundry detergents. Copyright © 2015 Elsevier B.V. All rights reserved.

  20. Validated stability-indicating methods for the determination of zafirlukast in the presence of its alkaline hydrolysis degradation product

    Directory of Open Access Journals (Sweden)

    Amal M. Abou Al Alamein

    2012-12-01

    Full Text Available Three simple stability-indicating methods for the analysis of Zafirlukast (ZAF in the presence of its alkaline degradation products were developed and validated as per the International Conference on Harmonization (ICH guidelines to evaluate the stability-indicating power of the proposed methods. The developed high-performance liquid chromatographic technique was achieved on ZORBAX–ODS (5 μm, 150 × 4.6 mm, i.d. by isocratic elution with a mixture of acetonitrile/0.05 M phosphate buffer, pH 5.0, (50:50; v/v as a mobile phase at flow rate of 1.0 mL min−1, followed by UV detection at 240 nm. The method could determine ZAF in the range of 2–40 μg mL−1 with a mean percentage recovery of 99.73 ± 0.903. The proposed HPLC method was utilized to investigate the kinetics of alkaline degradation of ZAF. First derivative of the ratio spectra (1DD method was applied to analyze the drug under investigation without any interference from its degradation product with a linearity range of 4–32 μg mL−1 and with a mean percentage recovery of 99.85 ± 0.608. A chemometric method was also developed using the partial least squares (PLS model for selective determination of ZAF in the range of 4–40 μg mL−1, the mean percentage recovery was found to be 100.00 ± 0.336.

  1. Pilot-Scale Production and Thermostability Improvement of the M23 Protease Pseudoalterin from the Deep Sea Bacterium Pseudoalteromonas sp. CF6-2

    Directory of Open Access Journals (Sweden)

    Jie Yang

    2016-11-01

    Full Text Available Pseudoalterin is the most abundant protease secreted by the marine sedimental bacterium Pseudoalteromonas sp. CF6-2 and is a novel cold-adapted metalloprotease of the M23 family. Proteases of the M23 family have high activity towards peptidoglycan and elastin, suggesting their promising biomedical and biotechnological potentials. To lower the fermentive cost and improve the pseudoalterin production of CF6-2, we optimized the fermentation medium by using single factor experiments, added 0.5% sucrose as a carbon source, and lowered the usage of artery powder from 1.2% to 0.6%. In the optimized medium, pseudoalterin production reached 161.15 ± 3.08 U/mL, 61% greater than that before optimization. We further conducted a small-scale fermentation experiment in a 5-L fermenter and a pilot-scale fermentation experiment in a 50-L fermenter. Pseudoalterin production during pilot-scale fermentation reached 103.48 ± 8.64 U/mL, 77% greater than that before the medium was optimized. In addition, through single factor experiments and orthogonal tests, we developed a compound stabilizer for pseudoalterin, using medically safe sugars and polyols. This stabilizer showed a significant protective effect for pseudoalterin against enzymatic thermal denaturation. These results lay a solid foundation for the industrial production of pseudoalterin and the development of its biomedical and biotechnological potentials.

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

    African Journals Online (AJOL)

    SERVER

    2008-04-17

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

  3. Hydrogen production from a rectangular horizontal filter press Divergent Electrode-Flow-Through (DEFT™) alkaline electrolysis stack

    Science.gov (United States)

    Gillespie, M. I.; Kriek, R. J.

    2017-12-01

    A membraneless Divergent Electrode-Flow-Through (DEFT™) alkaline electrolyser, for unlocking profitable hydrogen production by combining a simplistic, inexpensive, modular and durable design, capable of overcoming existing technology current density thresholds, is ideal for decentralised renewable hydrogen production, with the only requirement of electrolytic flow to facilitate high purity product gas separation. Scale-up of the technology was performed, representing a deviation from the original tested stack design, incorporating elongated electrodes housed in a filter press assembly. The pilot plant operating parameters were limited to a low flow velocity range (0.03 m s-1 -0.04 m s-1) with an electrode gap of 2.5 mm. Performance of this pilot plant demonstrated repeatability to results previously obtained. Mesh electrodes with geometric area of 344.32 cm2 were used for plant performance testing. A NiO anode and Ni cathode combination developed optimal performance yielding 508 mA cm-2 at 2 VDC in contrast to a Ni anode and cathode combination providing 467 mA cm-2 at 2.26 VDC at 0.04 m s-1, 30% KOH and 80 °C. An IrO2/RuO2/TiO2 anode and Pt cathode combination underwent catalyst deactivation. Owing to the nature of the gas/liquid separation system, gas qualities were inadequate compared to results achieved previously. Future improvements will provide qualities similar to results achieved before.

  4. Surface analysis and depth profiling of corrosion products formed in lead pipes used to supply low alkalinity drinking water.

    Science.gov (United States)

    Davidson, C M; Peters, N J; Britton, A; Brady, L; Gardiner, P H E; Lewis, B D

    2004-01-01

    Modern analytical techniques have been applied to investigate the nature of lead pipe corrosion products formed in pH adjusted, orthophosphate-treated, low alkalinity water, under supply conditions. Depth profiling and surface analysis have been carried out on pipe samples obtained from the water distribution system in Glasgow, Scotland, UK. X-ray diffraction spectrometry identified basic lead carbonate, lead oxide and lead phosphate as the principal components. Scanning electron microscopy/energy-dispersive x-ray spectrometry revealed the crystalline structure within the corrosion product and also showed spatial correlations existed between calcium, iron, lead, oxygen and phosphorus. Elemental profiling, conducted by means of secondary ion mass spectrometry (SIMS) and secondary neutrals mass spectrometry (SNMS) indicated that the corrosion product was not uniform with depth. However, no clear stratification was apparent. Indeed, counts obtained for carbonate, phosphate and oxide were well correlated within the depth range probed by SIMS. SNMS showed relationships existed between carbon, calcium, iron, and phosphorus within the bulk of the scale, as well as at the surface. SIMS imaging confirmed the relationship between calcium and lead and suggested there might also be an association between chloride and phosphorus.

  5. 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, Vladimí r; Stingl, Ulrich; Brune, Andreas

    2014-01-01

    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

  6. Oxidation of D-glucose and D-fructose with oxygen in aqueous, alkaline solutions. III. Kinetic approach to the product distribution

    NARCIS (Netherlands)

    de Wilt, H.G.J.; Kuster, Ben

    1972-01-01

    Based on a previously reported, integral reaction-scheme for the homogeneous oxidation of -glucose and -fructose with oxygen in aqueous, alkaline solutions, a kinetic model covering the product distribution has been developed. The model consists of a repeated set of reactions with constant rate

  7. Biomass production and energy source of thermophiles in a Japanese alkaline geothermal pool.

    Science.gov (United States)

    Kimura, Hiroyuki; Mori, Kousuke; Nashimoto, Hiroaki; Hattori, Shohei; Yamada, Keita; Koba, Keisuke; Yoshida, Naohiro; Kato, Kenji

    2010-02-01

    Microbial biomass production has been measured to investigate the contribution of planktonic bacteria to fluxations in dissolved organic matter in marine and freshwater environments, but little is known about biomass production of thermophiles inhabiting geothermal and hydrothermal regions. The biomass production of thermophiles inhabiting an 85 degrees C geothermal pool was measured by in situ cultivation using diffusion chambers. The thermophiles' growth rates ranged from 0.43 to 0.82 day(-1), similar to those of planktonic bacteria in marine and freshwater habitats. Biomass production was estimated based on cellular carbon content measured directly from the thermophiles inhabiting the geothermal pool, which ranged from 5.0 to 6.1 microg C l(-1) h(-1). This production was 2-75 times higher than that of planktonic bacteria in other habitats, because the cellular carbon content of the thermophiles was much higher. Quantitative PCR and phylogenetic analysis targeting 16S rRNA genes revealed that thermophilic H2-oxidizing bacteria closely related to Calderobacterium and Geothermobacterium were dominant in the geothermal pool. Chemical analysis showed the presence of H2 in gases bubbling from the bottom of the geothermal pool. These results strongly suggested that H2 plays an important role as a primary energy source of thermophiles in the geothermal pool.

  8. Production and partial characterization of alkaline feruloyl esterases by Fusarium oxysporum during submerged batch cultivation

    DEFF Research Database (Denmark)

    Topakas, E.; Christakopoulos, Paul

    2004-01-01

    Production of feruloyl esterases (FAEs) by Fusarium oxysporum was enhanced by optimization of initial pH of the culture medium, the type and concentration of nitrogen and carbon source. Submerged batch cultivation in a laboratory bioreactor (17 1) produced activity at 82 nkat g(-1) dry substrate....... Production of FAE does not therefore, require FA, however, production is diminished by the removal of esterified FA from the growth substrate. Optimal FAE activity was observed at pH 7 and 50 degreesC with 68 and 55% activity at pH 8 and pH 9, respectively. The esterase was fully stable at pH 5-8 and up...

  9. Biodiesel Production Using Waste Cooking Oil and Ethanol for Alkaline Catalysis

    OpenAIRE

    Bulla Pereira, Edwin A.; Sierra, Fabio E.; Guerrero, Carlos A.

    2014-01-01

    This work presents a study of the results of the project “Design of a Biodiesel Production Process Based on Cooking Oils at the Universidad Nacional de Colombia” (“Diseño de un proceso de producción de biodiesel a partir de aceites de fritura de la Universidad Nacional de Colombia”) carried out in 2013. Refined vegetable oils are the most commonly used to produce biodiesel fuels; however, used fried oils (auf from the Spanish acronym) make for a product with quality, yield and environmental b...

  10. Catalytic water co-existing with a product peptide in the active site of HIV-1 protease revealed by X-ray structure analysis.

    Directory of Open Access Journals (Sweden)

    Vishal Prashar

    Full Text Available BACKGROUND: It is known that HIV-1 protease is an important target for design of antiviral compounds in the treatment of Acquired Immuno Deficiency Syndrome (AIDS. In this context, understanding the catalytic mechanism of the enzyme is of crucial importance as transition state structure directs inhibitor design. Most mechanistic proposals invoke nucleophilic attack on the scissile peptide bond by a water molecule. But such a water molecule coexisting with any ligand in the active site has not been found so far in the crystal structures. PRINCIPAL FINDINGS: We report here the first observation of the coexistence in the active site, of a water molecule WAT1, along with the carboxyl terminal product (Q product peptide. The product peptide has been generated in situ through cleavage of the full-length substrate. The N-terminal product (P product has diffused out and is replaced by a set of water molecules while the Q product is still held in the active site through hydrogen bonds. The position of WAT1, which hydrogen bonds to both the catalytic aspartates, is different from when there is no substrate bound in the active site. We propose WAT1 to be the position from where catalytic water attacks the scissile peptide bond. Comparison of structures of HIV-1 protease complexed with the same oligopeptide substrate, but at pH 2.0 and at pH 7.0 shows interesting changes in the conformation and hydrogen bonding interactions from the catalytic aspartates. CONCLUSIONS/SIGNIFICANCE: The structure is suggestive of the repositioning, during substrate binding, of the catalytic water for activation and subsequent nucleophilic attack. The structure could be a snap shot of the enzyme active site primed for the next round of catalysis. This structure further suggests that to achieve the goal of designing inhibitors mimicking the transition-state, the hydrogen-bonding pattern between WAT1 and the enzyme should be replicated.

  11. Catalytic water co-existing with a product peptide in the active site of HIV-1 protease revealed by X-ray structure analysis.

    Science.gov (United States)

    Prashar, Vishal; Bihani, Subhash; Das, Amit; Ferrer, Jean-Luc; Hosur, Madhusoodan

    2009-11-17

    It is known that HIV-1 protease is an important target for design of antiviral compounds in the treatment of Acquired Immuno Deficiency Syndrome (AIDS). In this context, understanding the catalytic mechanism of the enzyme is of crucial importance as transition state structure directs inhibitor design. Most mechanistic proposals invoke nucleophilic attack on the scissile peptide bond by a water molecule. But such a water molecule coexisting with any ligand in the active site has not been found so far in the crystal structures. We report here the first observation of the coexistence in the active site, of a water molecule WAT1, along with the carboxyl terminal product (Q product) peptide. The product peptide has been generated in situ through cleavage of the full-length substrate. The N-terminal product (P product) has diffused out and is replaced by a set of water molecules while the Q product is still held in the active site through hydrogen bonds. The position of WAT1, which hydrogen bonds to both the catalytic aspartates, is different from when there is no substrate bound in the active site. We propose WAT1 to be the position from where catalytic water attacks the scissile peptide bond. Comparison of structures of HIV-1 protease complexed with the same oligopeptide substrate, but at pH 2.0 and at pH 7.0 shows interesting changes in the conformation and hydrogen bonding interactions from the catalytic aspartates. The structure is suggestive of the repositioning, during substrate binding, of the catalytic water for activation and subsequent nucleophilic attack. The structure could be a snap shot of the enzyme active site primed for the next round of catalysis. This structure further suggests that to achieve the goal of designing inhibitors mimicking the transition-state, the hydrogen-bonding pattern between WAT1 and the enzyme should be replicated.

  12. Effect of coppicing height on the regeneration and productivity of certain firewood shrubs in alkaline soils of north Indian plains

    Energy Technology Data Exchange (ETDEWEB)

    Misra, P.N.; Tewari, S.K.; Singh, Dheer; Katiyar, R.S. [National Botanical Research Inst., Lucknow (India)

    1995-12-31

    Four shrubs, viz. Hibiscus tiliaceus, Leucaena leucocephala, Vitex negundo and Sesbania sesban, were evaluated for their performance as firewood crops in coppiced stands of varying cutting heights (15, 30 and 45 cm) in repeated annual harvests (4) on alkaline soils of the North Indian plains. The dry wood yield of Leucaena and Sesbania ranged between 22.9-42.6 and 9.9-18.0 tonnes ha{sup -1} year{sup -1}, respectively. Leucaena showed progressive increases in yield from coppiced stumps up to the 4th harvest while Sesbania showed a reduction after the third harvest mainly due to the high degeneration (60%) of coppiced stumps. Degeneration was low (< 10%) in Leucaena and Vitex. The coppicing heights generally did not show any significant effect on the growth and productivity. The number of coppice shoots per stump increased with stump height and production of coppice shoots was maximum in Vitex and minimum in Leucaena. The average diameter of coppice shoots tended to decrease with increasing coppicing height of the stumps. (author)

  13. Chemical stabilization of cadmium in acidic soil using alkaline agronomic and industrial by-products.

    Science.gov (United States)

    Chang, Yao-Tsung; Hsi, Hsing-Cheng; Hseu, Zeng-Yei; Jheng, Shao-Liang

    2013-01-01

    In situ immobilization of heavy metals using reactive or stabilizing materials is a promising solution for soil remediation. Therefore, four agronomic and industrial by-products [wood biochar (WB), crushed oyster shell (OS), blast furnace slag (BFS), and fluidized-bed crystallized calcium (FBCC)] and CaCO3 were added to acidic soil (Cd = 8.71 mg kg(-1)) at the rates of 1%, 2%, and 4% and incubated for 90 d. Chinese cabbage (Brassica chinensis L.) was then planted in the soil to test the Cd uptake. The elevation in soil pH caused by adding the by-products produced a negative charge on the soil surface, which enhanced Cd adsorption. Consequently, the diethylenetriamine pentaacetic acid (DTPA)-extractable Cd content decreased significantly (P soil. These results from the sequential extraction procedure indicated that Cd converted from the exchangeable fraction to the carbonate or Fe-Mn oxide fraction. The long-term effectiveness of Cd immobilization caused by applying the 4 by-products was much greater than that caused by applying CaCO3. Plant shoot biomass clearly increased because of the by-product soil amendment. Cd concentration in the shoots was soil.

  14. Characterization of degradation products from alkaline wet oxidation of wheat straw

    DEFF Research Database (Denmark)

    Klinke, H.B.; Ahring, B.K.; Schmidt, A.S.

    2002-01-01

    to their chemical structure, e.g. diacids (oxalic and succinic acids), furan aldehydes, phenol aldehydes, phenol ketones and phenol acids. Aromatic aldehyde formation was correlated to severe conditions with high temperatures and low pH. Apart from CO2 and water, carboxylic acids were the main degradation products...... degreesC with addition of 12 bar oxygen and 6.5 g l(-1) Na2CO3. At these conditions the hemicellulose fraction from 100 g straw consisted of soluble hemicellulose (16 g), low molecular weight carboxylic acids (11 g), monomeric phenols (0.48 g) and 2-furoic acid (0.01 g). Formic acid and acetic acid...... constituted the majority of degradation products (8.5 g). The main phenol monomers were 4-hydroxybenzaldehyde, vanillin, syringaldehyde, acetosyringone (4-hydroxy-3,5-dimethoxy-acetophenone), vanillic acid and syringic acid, occurring in 0.04-0.12 g per 100 g straw concentrations. High lignin removal from...

  15. Improving the gas productivity of the alkaline electrolyzer through the circulation technique

    Directory of Open Access Journals (Sweden)

    Kitipong Tangphant

    2014-03-01

    Full Text Available This research aims to study and improve the efficiency of a KOH electrolyzer through the gas productivity of the electrolyzer with different the circulation technique. In this work, the conceptual design of an electrolyzer falls into 2 categories; without pumping and with pumping. Direct current electricity at 5 different levels of 10, 15, 20, 25 and 30 A are charged into the system and the gas flow rate generated from the electrolyzer is subsequently monitored. The results show that at 30 A the gas generated from the circulation with pumping and the circulation without pumping are 2.31 litre/min and 1.76 litre/min, respectively. It is also found that the energy consumed by both techniques is the same; however, the circulation with pumping design shows the better gas productivity than that of the circulation without pumping design.

  16. Reductive Alkaline Release of N-Glycans Generates a Variety of Unexpected, Useful Products.

    Science.gov (United States)

    Figl, Rudolf; Altmann, Friedrich

    2018-02-01

    Release of O-glycans by reductive β-elimination has become routine in many glyco-analytical laboratories and concomitant release of N-glycans has repeatedly been observed. Revisiting this somewhat forgotten mode of N-glycan release revealed that all kinds of N-glycans including oligomannosidic and complex-type N-glycans from plants with 3-linked fucose and from mammals with or without 6-linked fucose and with sialic acid could be recovered. However, the mass spectra of the obtained products revealed very surprising facts. Even after 16 h incubation in 1 M sodium borohydride, a large part of the glycans occurred in reducing form. Moreover, about one third emerged in the form of the stable amino-functionalized 1-amino-1-deoxy-glycitol. When avoiding acidic conditions, considerable amounts of glycosylamine were observed. In addition, a compound with a reduced asparagine and de-N-acetylation products, in particular of sialylated glycans, was seen. The relative yields of the products reducing glycosylamine, reducing N-glycan, 1-amino-1-deoxy-glycitol or glycitol could be controlled by the release conditions, foremost by temperature and borohydride concentration. Thus, chemical release of N-glycans constitutes a cost-saving alternative to enzymatic hydrolysis for the preparation of precursors for the production of reference compounds for various formats of N-glycan analysis. Moreover, it allows to obtain a stable amino-functionalized glycan derivative, which can be employed to construct glycan arrays or affinity matrices. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. A biotechnology perspective of fungal proteases

    Directory of Open Access Journals (Sweden)

    Paula Monteiro de Souza

    2015-06-01

    Full Text Available Proteases hydrolyze the peptide bonds of proteins into peptides and amino acids, being found in all living organisms, and are essential for cell growth and differentiation. Proteolytic enzymes have potential application in a wide number of industrial processes such as food, laundry detergent and pharmaceutical. Proteases from microbial sources have dominated applications in industrial sectors. Fungal proteases are used for hydrolyzing protein and other components of soy beans and wheat in soy sauce production. Proteases can be produced in large quantities in a short time by established methods of fermentation. The parameters such as variation in C/N ratio, presence of some sugars, besides several other physical factors are important in the development of fermentation process. Proteases of fungal origin can be produced cost effectively, have an advantage faster production, the ease with which the enzymes can be modified and mycelium can be easily removed by filtration. The production of proteases has been carried out using submerged fermentation, but conditions in solid state fermentation lead to several potential advantages for the production of fungal enzymes. This review focuses on the production of fungal proteases, their distribution, structural-functional aspects, physical and chemical parameters, and the use of these enzymes in industrial applications.

  18. Alkaline pretreatment of Mexican pine residues for bioethanol ...

    African Journals Online (AJOL)

    Alkaline pretreatment of Mexican pine residues for bioethanol production. ... Keywords: Lignocellulosic biomass, alkaline pretreatment, enzymatic hydrolysis, fermentable sugars, fermentation. African Journal of Biotechnology Vol. 12(31), pp.

  19. Production of class a biosolids with anoxic low dose alkaline treatment and odor management

    Energy Technology Data Exchange (ETDEWEB)

    Abu-Orf, M.M.; Brewster, J.; Oleszkiewicz, J.; Reimers, R.S.; Lagasse, P.; Amy, B.; Glindemann, D.

    2003-07-01

    The feasibility of full-scale anoxic disinfection of dewatered and digested sludge from Winnipeg, Manitoba with low lime doses and lagoon fly ash was investigated to determine if a class A product could be produced. Lime doses of 50g, 100g, and 200g per kg of biosolids (dry) were used along with fly ash doses of 500g. 1000g. and 1500g per kg of biosolids (dry). The mixed product was buried in eight-10 cubic meter trenches at the West End Water Pollution Control Center In Winnipeg. The trenches were backfilled with dirt and trapped to simulate anoxic conditions. Sampling cages were packed with the mixed product and pathogens non-indigenous to Winnipeg's biosolids. The cages were buried amongst the mixed biosolids in the trench. The non-indigenous pathogens spiked in the laboratory were the helminth Ascaris suum and the enteric virus reovirus. Samples were removed at days 12, 40, 69, 291, and 356 and were tested for the presence of fecal Coliform, Clostridium perfringens spores, Ascaris suum eggs, and reovirus. The pH, total solids, and free ammonia content of the mixed product were also determined for each sample. Odor was quantified for samples at both 291 and 356 days. Fecal Coliform bacteria and reovirus were completely inactivated for doses as low as 100g lime per kg biosolids (dry) and 50g lime + 500g fly ash per kg biosolids (dry). Spores of the bacteria C. perfringens experienced a 4-log reduction when treated with 100g lime per kg biosolids and a 5-log reduction when treated with doses as low as 50g lime + 500g fly ash per kg biosolids (dry) after 69 days. Ascaris eggs were completely inactivated in 5 gram packets for all treatments involving 100g lime per kg biosolids (dry) after 69 days. Class A pathogen requirements were met for all treatments involving a lime dose of at least 100g per kg biosolids. The odor potential from the produced biosolids is also assessed. (author)

  20. Mechanism of interleukin-13 production by granulocyte-macrophage colony-stimulating factor-dependent macrophages via protease-activated receptor-2.

    Science.gov (United States)

    Yamaguchi, Rui; Yamamoto, Takatoshi; Sakamoto, Arisa; Ishimaru, Yasuji; Narahara, Shinji; Sugiuchi, Hiroyuki; Hirose, Eiji; Yamaguchi, Yasuo

    2015-06-01

    Granulocyte-macrophage colony-stimulating factor (GM-CSF) promotes classically activated M1 macrophages. GM-CSF upregulates protease-activated receptor-2 (PAR-2) protein expression and activation of PAR-2 by human neutrophil elastase (HNE) regulates cytokine production. This study investigated the mechanism of PAR-2-mediated interleukin (IL)-13 production by GM-CSF-dependent macrophages stimulated with HNE. Adherent macrophages were obtained from primary cultures of human mononuclear cells. After stimulation with HNE to activate the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling pathway, IL-13 mRNA and protein levels were assessed by the reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. PAR-2 protein was detected in GM-CSF-dependent macrophages by Western blotting. Unexpectedly, PD98059 (an ERK1 inhibitor) increased IL-13 production, even at higher concentrations. Interestingly, U0126 (an ERK1/2 inhibitor) reduced IL-13 production in a concentration-dependent manner. Neither SB203580 (a p38alpha/p38beta inhibitor) nor BIRB796 (a p38gamma/p38delta inhibitor) affected IL-13 production, while TMB-8 (a calcium chelator) diminished IL-13 production. Stimulation with HNE promoted the production of IL-13 (a Th2 cytokine) by GM-CSF-dependent M1 macrophages. PAR-2-mediated IL-13 production may be dependent on the Ca(2+)/ERK2 signaling pathway. Copyright © 2015 Elsevier Inc. All rights reserved.

  1. Smart Methods for Linezolid Determination in the Presence of Alkaline and Oxidative Degradation Products Utilizing Their Overlapped Spectral Bands

    Science.gov (United States)

    Abd El-Monem Hegazy, M.; Shaaban Eissa, M.; Abd El-Sattar, O. I.; Abd El-Kawy, M. M.

    2014-09-01

    Linezolid (LIN) is considered the first available oxazolidinone antibacterial agent. It is susceptible to hydrolysis and oxidation. Five simple, accurate, sensitive and validated UV spectrophotometric methods were developed for LIN determination in the presence of its alkaline (ALK) and oxidative (OXD) degradation products in bulk powder and pharmaceutical formulation. Method A is a second derivative one (D2) in which LIN is determined at 240.9 nm. Method B is a pH-induced differential derivative one where LIN is determined using the fourth derivative (D4) of the difference spectra (ΔA) at 285.3 nm. Methods C, D, and E are manipulating ratio spectra, where C is the double divisor-ratio difference spectrophotometric one (DD-RD) in which LIN was determined by calculating the amplitude difference at 243.7 and 267.6 nm of the ratio spectra. Method D is the double divisor-first derivative of ratio spectra (DD-DD1) in which LIN was determined at 270.2 nm. Method E is a mean centering of ratio spectra one (MCR) in which LIN was determined at 318.0 nm. The developed methods have been validated according to ICH guidelines. The results were statistically compared to that of a reported HPLC method and there was no significant difference regarding both accuracy and precision.

  2. Green coconut mesocarp pretreated by an alkaline process as raw material for bioethanol production.

    Science.gov (United States)

    Soares, Jimmy; Demeke, Mekonnen M; Foulquié-Moreno, Maria R; Van de Velde, Miet; Verplaetse, Alex; Fernandes, Antonio Alberto Ribeiro; Thevelein, Johan M; Fernandes, Patricia Machado Bueno

    2016-09-01

    Cocos nucifera L., coconut, is a palm of high importance in the food industry, but a considerable part of the biomass is inedible. In this study, the pretreatment and saccharification parameters NaOH solution, pretreatment duration and enzyme load were evaluated for the production of hydrolysates from green coconut mesocarp using 18% (w/v) total solids (TS). Hydrolysates were not detoxified in order to preserve sugars solubilized during the pretreatment. Reduction of enzyme load from 15 to 7.5 filter paper cellulase unit (FPU)/g of biomass has little effect on the final ethanol titer. With optimized pretreatment and saccharification, hydrolysates with more than 7% (w/v) sugars were produced in 48h. Fermentation of the hydrolysate using industrial Saccharomyces cerevisiae strains produced 3.73% (v/v) ethanol. Our results showed a simple pretreatment condition with a high-solid load of biomass followed by saccharification and fermentation of undetoxified coconut mesocarp hydrolysates to produce ethanol with high titer. Copyright © 2016 Elsevier Ltd. All rights reserved.

  3. Cytomegalovirus protease targeted prodrug development.

    Science.gov (United States)

    Sabit, Hairat; Dahan, Arik; Sun, Jing; Provoda, Chester J; Lee, Kyung-Dall; Hilfinger, John H; Amidon, Gordon L

    2013-04-01

    Human cytomegalovirus (HCMV) is a prevalent virus that infects up to 90% of the population. The goal of this research is to determine if small molecular prodrug substrates can be developed for a specific HCMV encoded protease and thus achieve site-specific activation. HCMV encodes a 256 amino acid serine protease that is responsible for capsid assembly, an essential process for herpes virus production. The esterase activity of the more stable HCMV A143T/A144T protease mutant was evaluated with model p-nitrophenol (ONp) esters, Boc-Xaa-ONp (Ala, Leu, Ile, Val, Gln, Phe at the Xaa position). We demonstrate that the A143T/A144T mutant has esterase activity toward specific small ester compounds, e.g., Boc-L-Ala-ONp. Mono amino acid and dipeptide prodrugs of ganciclovir (GCV) were also synthesized and evaluated for hydrolysis by the A143T/A144T protease mutant in solution. Hydrolysis of these prodrugs was also evaluated in Caco-2 cell homogenates, human liver microsomes (HLMs), and rat and human plasma. For the selectivity potential of the prodrugs, the hydrolysis ratio was evaluated as a percentage of prodrug hydrolyzed by the HCMV protease over the percentages of prodrug hydrolyses by Caco-2 cell homogenates, HLMs, and human/rat plasma. A dipeptide prodrug of ganciclovir, Ac-l-Gln-l-Ala-GCV, emerged as a potential selective prodrug candidate. The results of this research demonstrate that targeting prodrugs for activation by a specific protease encoded by the infectious HCMV pathogen may be achievable.

  4. Improvement of shelf life of soymilk using immobilized protease of Oerskovia xanthineolytica NCIM 2839

    OpenAIRE

    Sahoo, A. K.; Gaikwad, V. S.; Ranveer, R. C.; Dandge, P. B.; Waghmare, S. R.

    2016-01-01

    Protease enzyme has lot of commercial applications, so the cost-effective production of protease using sunflower oil seed waste was carried out from Oerskovia xanthineolyitca NCIM 2839. The maximum protease production was after 24?h of incubation with 2.5?% oil seed waste concentration. O. xanthineolytica was found to produce two proteases?P1 and P2. The proteases were purified using 60?% cold acetone precipitation and DEAE-cellulose ion exchange chromatography. SDS-PAGE revealed molecular we...

  5. Advanced alkaline water electrolysis

    International Nuclear Information System (INIS)

    Marini, Stefania; Salvi, Paolo; Nelli, Paolo; Pesenti, Rachele; Villa, Marco; Berrettoni, Mario; Zangari, Giovanni; Kiros, Yohannes

    2012-01-01

    A short review on the fundamental and technological issues relevant to water electrolysis in alkaline and proton exchange membrane (PEM) devices is given. Due to price and limited availability of the platinum group metal (PGM) catalysts they currently employ, PEM electrolyzers have scant possibilities of being employed in large-scale hydrogen production. The importance and recent advancements in the development of catalysts without PGMs are poised to benefit more the field of alkaline electrolysis rather than that of PEM devices. This paper presents our original data which demonstrate that an advanced alkaline electrolyzer with performances rivaling those of PEM electrolyzers can be made without PGM and with catalysts of high stability and durability. Studies on the advantages/limitations of electrolyzers with different architectures do show how a judicious application of pressure differentials in a recirculating electrolyte scheme helps reduce mass transport limitations, increasing efficiency and power density.

  6. 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.

  7. Removal of fermentation inhibitors from alkaline peroxide pretreated and enzymatically hydrolyzed wheat straw: Production of butanol from hydrolysate using Clostridium beijerinckii in batch reactors

    International Nuclear Information System (INIS)

    Qureshi, Nasib; Saha, Badal C.; Hector, Ronald E.; Cotta, Michael A.

    2008-01-01

    In these studies, alkaline peroxide pretreatment of wheat straw was investigated. Pretreated wheat straw was hydrolyzed using cellulolytic and xylanolytic enzymes, and the hydrolysate was used to produce butanol using Clostridium beijerinckii P260. The culture produced less than 2.59 g L -1 acetone-butanol-ethanol (ABE) from alkaline peroxide wheat straw hydrolysate (APWSH) that had not been treated to reduce salt concentration (a neutralization product). However, fermentation was successful after inhibitors (salts) were removed from the hydrolysate by electrodialysis. A control glucose fermentation resulted in the production of 21.37 g L -1 ABE, while salt removed APWSH resulted in the production of 22.17 g L -1 ABE. In the two fermentations, reactor productivities were 0.30 and 0.55 g L -1 h -1 , respectively. A comparison of use of different substrates (corn fiber, wheat straw) and different pretreatment techniques (dilute sulfuric acid, alkaline peroxide) suggests that generation of inhibitors is substrate and pretreatment specific

  8. Enhancement in production of recombinant two-chain Insulin Glargine by over-expression of Kex2 protease in Pichia pastoris.

    Science.gov (United States)

    Sreenivas, Suma; Krishnaiah, Sateesh M; Govindappa, Nagaraja; Basavaraju, Yogesh; Kanojia, Komal; Mallikarjun, Niveditha; Natarajan, Jayaprakash; Chatterjee, Amarnath; Sastry, Kedarnath N

    2015-01-01

    Glargine is an analog of Insulin currently being produced by recombinant DNA technology using two different hosts namely Escherichia coli and Pichia pastoris. Production from E. coli involves the steps of extraction of inclusion bodies by cell lysis, refolding, proteolytic cleavage and purification. In P. pastoris, a single-chain precursor with appropriate disulfide bonding is secreted to the medium. Downstream processing currently involves use of trypsin which converts the precursor into two-chain final product. The use of trypsin in the process generates additional impurities due to presence of Lys and Arg residues in the Glargine molecule. In this study, we describe an alternate approach involving over-expression of endogenous Kex2 proprotein convertase, taking advantage of dibasic amino acid sequence (Arg-Arg) at the end of B-chain of Glargine. KEX2 gene over-expression in Pichia was accomplished by using promoters of varying strengths to ensure production of greater levels of fully functional two-chain Glargine product, confirmed by HPLC and mass analysis. In conclusion, this new production process involving Kex2 protease over-expression improves the downstream process efficiency, reduces the levels of impurities generated and decreases the use of raw materials.

  9. Sulfur-oxidizing autotrophic and mixotrophic denitrification processes for drinking water treatment: elimination of excess sulfate production and alkalinity requirement.

    Science.gov (United States)

    Sahinkaya, Erkan; Dursun, Nesrin

    2012-09-01

    This study evaluated the elimination of alkalinity need and excess sulfate generation of sulfur-based autotrophic denitrification process by stimulating simultaneous autotrophic and heterotrophic (mixotrophic) denitrification process in a column bioreactor by methanol supplementation. Also, denitrification performances of sulfur-based autotrophic and mixotrophic processes were compared. In autotrophic process, acidity produced by denitrifying sulfur-oxidizing bacteria was neutralized by the external NaHCO(3) supplementation. After stimulating mixotrophic denitrification process, the alkalinity need of the autotrophic process was satisfied by the alkalinity produced by heterotrophic denitrifiers. Decreasing and lastly eliminating the external alkalinity supplementation did not adversely affect the process performance. Complete denitrification of 75 mg L(-1) NO(3)-N under mixotrophic conditions at 4 h hydraulic retention time was achieved without external alkalinity supplementation and with effluent sulfate concentration lower than the drinking water guideline value of 250 mg L(-1). The denitrification rate of mixotrophic process (0.45 g NO(3)-N L(-1) d(-1)) was higher than that of autotrophic one (0.3 g NO(3)-N L(-1) d(-1)). Batch studies showed that the sulfur-based autotrophic nitrate reduction rate increased with increasing initial nitrate concentration and transient accumulation of nitrite was observed. Copyright © 2012 Elsevier Ltd. All rights reserved.

  10. 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. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Full-scale production of VFAs from sewage sludge by anaerobic alkaline fermentation to improve biological nutrients removal in domestic wastewater.

    Science.gov (United States)

    Liu, He; Han, Peng; Liu, Hongbo; Zhou, Guangjie; Fu, Bo; Zheng, Zhiyong

    2018-07-01

    A full-scale project of thermal-alkaline pretreatment and alkaline fermentation of sewage sludge was built to produce volatile fatty acids (VFAs) which was then used as external carbon source for improving biological nitrogen and phosphorus removals (BNPR) in wastewater plant. Results showed this project had efficient and stable performances in VFA production, sludge reduce and BNPR. Hydrolysis rate in pretreatment, VFAs yield in fermentation and total VS reduction reached 68.7%, 261.32 mg COD/g VSS and 54.19%, respectively. Moreover, fermentation liquid with VFA presented similar efficiency as acetic acid in enhancing BNPR, obtaining removal efficiencies of nitrogen and phosphorus up to 72.39% and 89.65%, respectively. Finally, the project also presented greater economic advantage than traditional processes, and the net profits for VFAs and biogas productions are 9.12 and 3.71 USD/m 3 sludge, respectively. Long-term operation indicated that anaerobic alkaline fermentation for VFAs production is technically and economically feasible for sludge carbon recovery. Copyright © 2018 Elsevier Ltd. All rights reserved.

  12. A novel process for volatile fatty acids production from syngas by integrating with mesophilic alkaline fermentation of waste activated sludge

    DEFF Research Database (Denmark)

    Rao, Yue; Wan, Jingjing; Liu, Yafeng

    2018-01-01

    The present study proposed and demonstrated a novel process for the bioconversion of syngas (mainly CO and H2) to valuable volatile fatty acids (VFA) by integrating with mesophilic alkaline fermentation of waste activated sludge (WAS). The results showed that although pH 9 was suitable for VFA...... to the mesophilic alkaline fermentation of WAS at pH 10 not only resulted in the enrichment of some known bacteria related with syngas conversion, but also changed the microbial community compositions for the fermentation of WAS....

  13. Exogenous protease supplementation of poultry by-product meal-based diets for broilers: Effects on growth, carcass characteristics and nutrient digestibility.

    Science.gov (United States)

    Mahmood, T; Mirza, M A; Nawaz, H; Shahid, M

    2018-02-01

    An experiment was conducted to investigate the effects of three levels (0%, 3% and 6%) of poultry by-product meal (PBM) with or without protease on broiler growth, carcass characteristics and nutrient digestibility from 1 to 35 days. Two hundred and forty birds (n = 240) were fed equi-caloric and equi-nitrogenous (ME 2850 kcal/kg; CP 20%) diets throughout the experiment. The enzyme supplementation increased feed intake (p  .05) from 1 to 21 days. Increasing level of PBM decreased feed intake (p < .05), but body weight gain was improved (p < .05) at 3% PBM level during 1 to 21 days. The feed:gain was improved (p < .05) in birds fed diets containing 3% PBM. The feed:gain was also improved in birds fed diets containing 3% PBM from 1 to 35 days. However, feed intake and body weight gain in birds fed diets containing PBM remained unaffected. An interaction (p < .01) on feed intake between enzyme and PBM was noticed during 1 to 21 days. However, no interaction was recorded for body weight gain and feed:gain. The per cent carcass yield improved (p < .01) in birds fed diets supplemented with enzyme. The per cent breast meat yield was depressed (p < .005) in birds fed diets containing PBM. Apparent metabolizable energy (p < .001), nitrogen retention (p < .01), apparent metabolizable energy corrected for nitrogen (p < .001), and apparent digestibility coefficient for nitrogen (p < .01) improved in birds fed diets containing enzyme; however, a reverse was noticed in those fed diets containing only PBM. In conclusion, inclusion of 3% PBM along with supplementation of exogenous protease improved performance and nutrient digestibility in broilers. © 2017 Blackwell Verlag GmbH.

  14. Escherichia coli contains a soluble ATP-dependent protease (Ti) distinct from protease La

    Energy Technology Data Exchange (ETDEWEB)

    Hwang, B.J.; Park, W.J.; Chung, C.H.; Goldberg, A.L.

    1987-08-01

    The energy requirement for protein breakdown in Escherichia coli has generally been attributed to the ATP-dependence of protease La, the lon gene product. The authors have partially purified another ATP-dependent protease from lon/sup -/ cells that lack protease La (as shown by immunoblotting). This enzyme hydrolyzes (/sup 3/H)methyl-casein to acid-soluble products in the presence of ATP and Mg/sup 2 +/. ATP hydrolysis appears necessary for proteolytic activity. Since this enzyme is inhibited by diisopropyl fluorophosphate, it appears to be a serine protease, but it also contains essential thiol residues. They propose to name this enzyme protease Ti. It differs from protease La in nucleotide specificity, inhibitor sensitivity, and subunit composition. On gel filtration, protease Ti has an apparent molecular weight of 370,000. It can be fractionated by phosphocellulose chromatography or by DEAE chromatography into two components with apparent molecular weights of 260,000 and 140,000. When separated, they do not show preteolytic activity. One of these components, by itself, has ATPase activity and is labile in the absence of ATP. The other contains the diisopropyl fluorophosphate-sensitive proteolytic site. These results and the similar findings of Katayama-Fujimura et al. indicate that E. coli contains two ATP-hydrolyzing proteases, which differ in many biochemical features and probably in their physiological roles.

  15. Escherichia coli contains a soluble ATP-dependent protease (Ti) distinct from protease La

    International Nuclear Information System (INIS)

    Hwang, B.J.; Park, W.J.; Chung, C.H.; Goldberg, A.L.

    1987-01-01

    The energy requirement for protein breakdown in Escherichia coli has generally been attributed to the ATP-dependence of protease La, the lon gene product. The authors have partially purified another ATP-dependent protease from lon - cells that lack protease La (as shown by immunoblotting). This enzyme hydrolyzes [ 3 H]methyl-casein to acid-soluble products in the presence of ATP and Mg 2+ . ATP hydrolysis appears necessary for proteolytic activity. Since this enzyme is inhibited by diisopropyl fluorophosphate, it appears to be a serine protease, but it also contains essential thiol residues. They propose to name this enzyme protease Ti. It differs from protease La in nucleotide specificity, inhibitor sensitivity, and subunit composition. On gel filtration, protease Ti has an apparent molecular weight of 370,000. It can be fractionated by phosphocellulose chromatography or by DEAE chromatography into two components with apparent molecular weights of 260,000 and 140,000. When separated, they do not show preteolytic activity. One of these components, by itself, has ATPase activity and is labile in the absence of ATP. The other contains the diisopropyl fluorophosphate-sensitive proteolytic site. These results and the similar findings of Katayama-Fujimura et al. indicate that E. coli contains two ATP-hydrolyzing proteases, which differ in many biochemical features and probably in their physiological roles

  16. Biological activity of egg-yolk protein by-product hydrolysates obtained with the use of non-commercial plant protease

    Directory of Open Access Journals (Sweden)

    A. Zambrowicz

    2015-12-01

    Full Text Available Enzymatic hydrolysis leads to improved functional and biological properties of protein by-products, which can be further used as nutraceuticals and protein ingredients for food applications.The present study evaluated ACE-inhibitory, antioxidant and immunostimulating activities in hydrolysates of egg-yolk protein by-product (YP, generated during industrial process of delipidation of yolk. The protein substrate was hydrolyzed using non-commercial protease from Asian pumpkin (Cucurbita ficifolia. The reaction was conducted in 0.1 M Tris-HCl buffer (pH 8.0 at temperature of 37°C for 4 hours using different enzyme doses (100-1000 U/mg of substrate. The protein degradation was monitored by the determination of the degree of hydrolysis (DH, release of free amino groups (FAG and by RP-HPLC. In the obtained hydrolysates we also evaluated biological activities. It was shown that the highest DH of substrate (46.6% was obtained after 4h of reaction at the highest amount of enzyme. This hydrolysate exhibited antioxidant activity, including ferricion reducing (FRAP (56.41 μg Fe2+/mg, ferric ion chelating (695.76 μg Fe2+/mg and DPPH free radical scavenging (0.89 μmol troloxeq/mg as well as ACE-inhibitory (IC50=837.75 μg/mL activities.The research showed improved biological properties of enzymatically modified YP by-product.

  17. 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.

  18. The alkaline comet assay used in evaluation of genotoxic damage of drinking water disinfection by-products (bromoform and chloroform

    Directory of Open Access Journals (Sweden)

    Messaouda Khallef

    2015-06-01

    Full Text Available The alkaline comet assay (pH 12.3 is a useful method for monitoring genotoxic effects of environmental pollutants in the root nuclei of Allium cepa and various plants; it allows the detection of single- and double-strand breaks, incomplete excision-repair sites and cross-links. It has been introduced to detect even small changes in DNA structure. It is a technically simple, highly sensitive, fast and economic test which detects in vitro and in vivo genotoxicity (DNA integrity and packing mode in any cell types examined, and requires just a few cells for its execution (Liman et al., 2011; Yıldız et al., 2009. Chloroform and bromoform are the most important trihalomethanes found in drinking water. Different concentrations of bromoform (25, 50, 75and 100µg/ml and chloroform (25, 50, 100 and 200 µg/ml were introduced to onion tuber roots. Distilled water was used as a negative control and methyl methansulfonate (MMS-10 µg/ml as positive control. All obtained data were subjected to statistical analyses by using SPSS 15.0 for Windows software. For comparison purposes, Duncan multiple range tests using one-way analysis of variance (ANOVA were employed and p<0.05 was accepted as the test of significance. Comet assay results showed that DNA damage was significant at p <0.05 for the different concentrations of chloroform and bromoform compared to the negative control which has a damage rate equal to 3.5 ± 0.7 and the positive control which has damage rate equal to 13.5 ± 2.12. The exposure of root tip cells to these disinfection by-products increases DNA damage. All concentrations examined in this study of bromoform and chloroform cause significant harm, which could be due to DNA damage induced by oxidative stress. The measurement of DNA damage in the nuclei of higher plant tissues is a new area of study with SCGE. This assay could be incorporated into in situ monitoring of atmosphere, water and soil: the comet assay allows a fast detection without

  19. Bacterial proteases and virulence

    DEFF Research Database (Denmark)

    Frees, Dorte; Brøndsted, Lone; Ingmer, Hanne

    2013-01-01

    signalling to short-circuit host cell processes. Common to both intra- and extracellular proteases is the tight control of their proteolytic activities. In general, substrate recognition by the intracellular proteases is highly selective which is, in part, attributed to the chaperone activity associated...... tolerance to adverse conditions such as those experienced in the host. In the membrane, HtrA performs similar functions whereas the extracellular proteases, in close contact with host components, pave the way for spreading infections by degrading host matrix components or interfering with host cell...... with the proteases either encoded within the same polypeptide or on separate subunits. In contrast, substrate recognition by extracellular proteases is less selective and therefore these enzymes are generally expressed as zymogens to prevent premature proteolytic activity that would be detrimental to the cell...

  20. 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.

  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. Changes in protein metabolism after irradiation. Pt. 1. Protease activity, protease pattern, protein and free amino acids in cytoplasm and cell organelles of the rat spleen after 600 R whole body x irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Valet, G [Max-Planck-Institut fuer Biochemie, Muenchen (F.R. Germany). Abt. fuer Experimentelle Medizin

    1975-12-01

    The protease activity of cytoplasm and cell organelles of the rat spleen against spleen protein and hemoglobin as a substrate increases during a initial reaction phase of the organism on the first day after 600 R whole body X-irradiation. The alkaline protease in the cytoplasm and the acid protease in the cell organelles increase, whereas the protease activity against externally added hemoglobin as substrate decreases below the initial values. The protein, the protease activity and the free amino acids of the cytoplasm and the cell organelles decrease during the disease phase on day 3 and 4 after irradiation. The protein loss of the spleen is therefore not explained by an increased protease activity. Acid proteases appear in the cytoplasm which derive probably from the cell organelles. The protease activity and the free amino acids are increased in the cytoplasm and the cell organelles during the regeneration phase of the organism between day 15 and 18 after irradiation.

  3. Asparagus stem as a new lignocellulosic biomass feedstock for anaerobic digestion: increasing hydrolysis rate, methane production and biodegradability by alkaline pretreatment.

    Science.gov (United States)

    Chen, Xiaohua; Gu, Yu; Zhou, Xuefei; Zhang, Yalei

    2014-07-01

    Recently, anaerobic digestion of lignocellulosic biomass for methane production has attracted considerable attention. However, there is little information regarding methane production from asparagus stem, a typical lignocellulosic biomass, by anaerobic digestion. In this study, alkaline pretreatment of asparagus stem was investigated for its ability to increase hydrolysis rate and methane production and to improve biodegradability (BD). The hydrolysis rate increased with increasing NaOH dose, due to higher removal rates of lignin and hemicelluloses. However, the optimal NaOH dose was 6% (w/w) according to the specific methane production (SMP). Under this condition, the SMP and the technical digestion time of the NaOH-treated asparagus stem were 242.3 mL/g VS and 18 days, which were 38.4% higher and 51.4% shorter than those of the untreated sample, respectively. The BD was improved from 40.1% to 55.4%. These results indicate that alkaline pretreatment could be an efficient method for increasing methane production from asparagus stem. Copyright © 2014 Elsevier Ltd. All rights reserved.

  4. Recovery of vanadium (V) from spent catalysts used in sulfuric acid production units by acid or alkaline leaching

    International Nuclear Information System (INIS)

    Abdulbaki, M.; Stas, J.; Shino, O.; Asaad, K.; Al-Kassemi, H.; Al-Qabani, F.

    2008-01-01

    The present paper, studies the recovery of vanadium from the spent catalyst by using acidic or alkaline leaching technique. The optimal conditions of spent catalyst leaching have been studied. It has been shown that 20%(w/w) of sulfuric acid is the most suitable for leaching process at 70 Centigrade. The precipitation of vanadium using some alkaline media (Na 2 CO 3 , (NH 4 )CO 3 and NH 4 OH) has been also studied, it has been shown that ammonium hydroxide was the best at 60 degree, and iron was co-precipitated with vanadium which pollute the obtained red cake. So it is necessary to use liquid-liquid extraction technique for the separation between vanadium and iron and to have iron free red cake. (author)

  5. Hydrothermal and alkaline hydrothermal pretreatments plus anaerobic digestion of sewage sludge for dewatering and biogas production: Bench-scale research and pilot-scale verification.

    Science.gov (United States)

    Li, Chunxing; Wang, Xingdong; Zhang, Guangyi; Yu, Guangwei; Lin, Jingjiang; Wang, Yin

    2017-06-15

    To test the feasibility and practicability of the process combing hydrothermal pretreatment for dewatering with biogas production for full utilization of sewage sludge, hydrothermal/alkaline hydrothermal pretreatments and in turn anaerobic digestion of the filtrates obtained after dewatering the pretreated sludge were performed at bench- and pilot-scales. The hydrothermal temperature fell within the range of 140 °C-220 °C and the pretreatment time varied from 30 min to 120 min. For the alkaline hydrothermal pretreatment the pH value of the sludge was adjusted to 9.0-11.0 by adding Ca(OH) 2 . The results showed that the dewaterability of the sewage sludge was improved with increasing pretreatment temperature but the impact of the pretreatment time was not significant. The addition of Ca(OH) 2 gave better performance on the subsequent mechanical dewatering of the pretreated sludge compared to pure hydrothermal pretreatment, and the higher the pH value was, the better the dewaterability of the pretreated sludge was. The conditions of 180 °C/30 min and 160 °C/60 min/pH = 10.0 (for hydrothermal and alkaline hydrothermal pretreatments, respectively) resulted in relatively good results in the theoretical energy balance, which were verified in the pilot-scale tests. Based on the data from the pilot tests, the alkaline hydrothermal process realized self-sufficiency in energy at the cost of a proper amount of CaO. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. PARTIAL PURIFICATION AND CHARACTERIZATION OF ALKALOPHILIC PROTEASE FROM PSEUDOMONAS AERUGINOSA

    Directory of Open Access Journals (Sweden)

    R. Satheeskumar

    2013-10-01

    Full Text Available Partial purification and characterization of alkalophilic protease production from Pseudomonas aeruginosa was isolated from the gut of marine and coastal waters shrimp Penaeus monodon. The protease production was assayed in submerged fermentation to produce maximum protease activity (423 ± 0.09 U/ml. The enzyme was precipitated with ammonium sulphate and partially purified by ion exchange chromatography through DEAE Sephadex A-50 column. In 10th fraction showed maximum protease activity (734 ± 0.18 U/ml with increase in purification fold. The molecular weight of protease from Pseudomonas aeruginosa was recorded as 60 kDa. The stability of protease was tested at various pH and temperature; it showed maximum protease activity at pH-9 and temperature 50ºC. Among the various surfactants tested for enzyme stability, maximum activity was retained in poly ethylene glycol. The compatibility of protease enzyme with various commercial detergents; the enzyme retained maximum protease activity in tide. The results are indicated that all these properties make the bacterial proteases are most suitable for wide industrial applications.

  7. Quantitative evaluation of the alkaline phosphatase activity in industrial and traditional dairy products supplied in Ahvaz as an indicator of pasteurization

    Directory of Open Access Journals (Sweden)

    M. Zarei

    2017-05-01

    Full Text Available Alkaline phosphatase is an indigenous milk enzyme and is probably, the most important indigenous milk enzyme from a dairy technology viewpoint which is used to determine the efficacy of the pasteurization process. The aim of this study was to assess the alkaline phosphatase activity of 200 samples of industrial and traditional yoghurt, ice cream and cheese, as well as raw and pasteurized milk samples. To achieve this purpose, p-nitrophenylphosphate was used as substrate and the amount of liberated p-nitrophenol was measured spectrophotometrically. The amount of liberated p-nitrophenol in all samples of raw milk was very high (6839±4070 µg/ml but in pasteurized milk samples, the amount was in the range of 0.75-52.96 µg/ml and 88% of the samples had less than 10 µg p-nitrophenol/ml, the maximum permissible limit of p-nitrophenol in pasteurized products. The amount of liberated p-nitrophenol was in the range of 5.68-1210 µg/ml and 2.61-18.22 µg/ml in traditional and industrial cheese samples, respectively and it was estimated at the range of 0.75-26.67 µg/ml and 0.71- 35.82 µg/ml for traditional and industrial ice cream samples, respectively. The lowest alkaline phosphatase activity was observed in both industrial and traditional yoghurt samples. Meanwhile, p-nitrophenol in 12% of industrial cheese, 44% of traditional cheese and 16% of both industrial and traditional ice cream samples was higher than 10 µg/ml which could be due to the inadequate pasteurization of the product or cross contamination with raw milk. The results of the present study showed a need for more strict attention in the pasteurization of milk and its products.

  8. Diallylsulfide attenuates excessive collagen production and apoptosis in a rat model of bleomycin induced pulmonary fibrosis through the involvement of protease activated receptor-2

    Energy Technology Data Exchange (ETDEWEB)

    Kalayarasan, Srinivasan, E-mail: kalaivasanbio@gmail.com; Sriram, Narayanan; Soumyakrishnan, Syamala; Sudhandiran, Ganapasam, E-mail: sudhandiran@yahoo.com

    2013-09-01

    Pulmonary fibrosis (PF) can be a devastating lung disease. It is primarily caused by inflammation leading to severe damage of the alveolar epithelial cells. The pathophysiology of PF is not yet been clearly defined, but studying lung parenchymal injury by involving reactive oxygen species (ROS) through the activation of protease activated receptor-2 (PAR-2) may provide promising results. PAR-2 is a G-protein coupled receptor is known to play an important role in the development of PF. In this study, we investigated the inhibitory role of diallylsulfide (DAS) against ROS mediated activation of PAR-2 and collagen production accompanied by epithelial cell apoptosis. Bleomycin induced ROS levels may prompt to induce the expression of PAR-2 as well as extracellular matrix proteins (ECM), such as MMP 2 and 9, collagen specific proteins HSP-47, α-SMA, and cytokines IL-6, and IL-8RA. Importantly DAS treatment effectively decreased the expression of all these proteins. The inhibitory effect of DAS on profibrotic molecules is mediated by blocking the ROS level. To identify apoptotic signaling as a mediator of PF induction, we performed apoptotic protein expression, DNA fragmentation analysis and ultrastructural details of the lung tissue were performed. DAS treatment restored all these changes to near normalcy. In conclusion, treatment of PF bearing rats with DAS results in amelioration of the ROS production, PAR-2 activation, ECM production, collagen synthesis and alveolar epithelial cell apoptosis during bleomycin induction. We attained the first evidence that treatment of DAS decreases the ROS levels and may provide a potential therapeutic effect attenuating bleomycin induced PF. - Highlights: • DAS inhibits PAR-2 activity; bleomycin stimulates PAR-2 activity. • Increase in PAR-2 activity is correlated with pulmonary fibrosis • DAS reduces pro-inflammatory activity linked to facilitating pulmonary fibrosis. • DAS inhibits apoptosis of alveolar epithelial cells.

  9. Diallylsulfide attenuates excessive collagen production and apoptosis in a rat model of bleomycin induced pulmonary fibrosis through the involvement of protease activated receptor-2

    International Nuclear Information System (INIS)

    Kalayarasan, Srinivasan; Sriram, Narayanan; Soumyakrishnan, Syamala; Sudhandiran, Ganapasam

    2013-01-01

    Pulmonary fibrosis (PF) can be a devastating lung disease. It is primarily caused by inflammation leading to severe damage of the alveolar epithelial cells. The pathophysiology of PF is not yet been clearly defined, but studying lung parenchymal injury by involving reactive oxygen species (ROS) through the activation of protease activated receptor-2 (PAR-2) may provide promising results. PAR-2 is a G-protein coupled receptor is known to play an important role in the development of PF. In this study, we investigated the inhibitory role of diallylsulfide (DAS) against ROS mediated activation of PAR-2 and collagen production accompanied by epithelial cell apoptosis. Bleomycin induced ROS levels may prompt to induce the expression of PAR-2 as well as extracellular matrix proteins (ECM), such as MMP 2 and 9, collagen specific proteins HSP-47, α-SMA, and cytokines IL-6, and IL-8RA. Importantly DAS treatment effectively decreased the expression of all these proteins. The inhibitory effect of DAS on profibrotic molecules is mediated by blocking the ROS level. To identify apoptotic signaling as a mediator of PF induction, we performed apoptotic protein expression, DNA fragmentation analysis and ultrastructural details of the lung tissue were performed. DAS treatment restored all these changes to near normalcy. In conclusion, treatment of PF bearing rats with DAS results in amelioration of the ROS production, PAR-2 activation, ECM production, collagen synthesis and alveolar epithelial cell apoptosis during bleomycin induction. We attained the first evidence that treatment of DAS decreases the ROS levels and may provide a potential therapeutic effect attenuating bleomycin induced PF. - Highlights: • DAS inhibits PAR-2 activity; bleomycin stimulates PAR-2 activity. • Increase in PAR-2 activity is correlated with pulmonary fibrosis • DAS reduces pro-inflammatory activity linked to facilitating pulmonary fibrosis. • DAS inhibits apoptosis of alveolar epithelial cells

  10. Exceptional enhancement of H{sub 2} production in alkaline environment over plasmonic Au/TiO{sub 2} photocatalyst under visible light

    Energy Technology Data Exchange (ETDEWEB)

    Meng, Xianguang; Liu, Guigao [Graduate School of Chemical Science and Engineering, Hokkaido University, Sapporo 060-0814 (Japan); Environmental Remediation Materials Unit and International Center for Materials Nanoarchitectonics (WPI-MANA), 1-1 Namiki, Tsukuba, Ibaraki 305-0044 (Japan); Yu, Qing [Graduate School of Chemical Science and Engineering, Hokkaido University, Sapporo 060-0814 (Japan); Wang, Tao; Chang, Kun; Li, Peng [Environmental Remediation Materials Unit and International Center for Materials Nanoarchitectonics (WPI-MANA), 1-1 Namiki, Tsukuba, Ibaraki 305-0044 (Japan); Liu, Lequan, E-mail: Jinhua.YE@nims.go.jp, E-mail: Lequan.Liu@tju.edu.cn [TU-NIMS Joint Research Center, School of Materials Science and Engineering, Tianjin University, 92 Weijin Road, Tianjin 300072 (China); Ye, Jinhua, E-mail: Jinhua.YE@nims.go.jp, E-mail: Lequan.Liu@tju.edu.cn [Graduate School of Chemical Science and Engineering, Hokkaido University, Sapporo 060-0814 (Japan); Environmental Remediation Materials Unit and International Center for Materials Nanoarchitectonics (WPI-MANA), 1-1 Namiki, Tsukuba, Ibaraki 305-0044 (Japan); TU-NIMS Joint Research Center, School of Materials Science and Engineering, Tianjin University, 92 Weijin Road, Tianjin 300072 (China)

    2015-10-01

    A reaction environment modulation strategy was employed to promote the H{sub 2} production over plasmonic Au/semiconductor composites. It is shown that the fast consumption of the holes in plasmonic Au nanoparticles by methanol in alkaline reaction environment remarkably increases H{sub 2} generation rate under visible light. The photocatalytic reaction is mainly driven by the interband transition of plasmonic Au nanoparticles, and the apparent quantum efficiency of plasmon-assisted H{sub 2} production at pH 14 reaches 6% at 420 nm. The reaction environment control provides a simple and effective way for the highly efficient solar fuel production from biomass reforming through plasmonic photocatalysis in future.

  11. Partial characterisation of digestive proteases of the Mayan cichlid Cichlasoma urophthalmus.

    Science.gov (United States)

    Cuenca-Soria, C A; Álvarez-González, C A; Ortiz-Galindo, J L; Nolasco-Soria, H; Tovar-Ramírez, D; Guerrero-Zárate, R; Castillo-Domínguez, A; Perera-García, M A; Hernández-Gómez, R; Gisbert, E

    2014-06-01

    The characterisation of digestive proteases in native freshwater fish such as the Mayan cichlid Cichlasoma urophthalmus provides scientific elements that may be used to design balanced feed that matches with the digestive capacity of the fish. The purpose of this study was to characterise the digestive proteases, including the effect of the pH and the temperature on enzyme activity and stability, as well as the effect of inhibitors using multienzymatic extracts of the stomach and intestine of C. urophthalmus juveniles. Results showed that the optimum activities of the acid and alkaline proteases occurred at pH values of 3 and 9, respectively, whereas their optimum temperatures were 55 and 65 °C, respectively. The acid proteases were most stable at pH values of 2–3 and at temperatures of 35–45 °C, whereas the alkaline proteases were most stable at pH values of 6–9 and at 25–55 °C. The inhibition assays recorded a residual activity of 4% with pepstatin A for the acid proteases. The inhibition of the alkaline proteases was greater than 80% with TPCK, TLCK, EDTA and ovalbumin, and of 60 and 43.8% with PMSF and SBT1, respectively. The results obtained in this study make it possible to state that C. urophthalmus has a sufficiently complete digestive enzyme machinery to degrade food items characteristic of an omnivorous fish species, although specimens showed a tendency to carnivory.

  12. Production and Optimization of Alkaline Lipase by a Novel Psychrotolerant and Halotolerant Strain Planomicrobium okeanokoites ABN-IAUF-2 Isolated from Persian Gulf

    Directory of Open Access Journals (Sweden)

    Elham Akbari

    2016-04-01

    Full Text Available lipases have many different applications in detergents, cleaners, food industry, pharmaceutical industry, pulp and paper production and leather industry, but the extremophile lipases have more range applications. These enzymes are resistant to the high salty, temperature and alkaline conditions. Halophiles were isolated from Persian Gulf, Iran at 20°C in the presence of 10% NaCl. For screening the lipase producing bacteria, Rhodamine B agar and minimal medium were used. Then in order to find the best growth condition for the production of lipase, singlefactor optimization was carried out. The best environmental conditions and their interactions for lipase production were obtained using 16 levels Taguchi statistical test. The WS4 isolate indicated a good lipase activity. The 16srDNA sequencing revealed that the WS4 isolate was Planomicrobium okeanokoites. We named this novel strain Planomicrobium okeanokoites ABN-IAUF-2 and its 16s-rDNA sequence was deposited in GenBank, NCBI, under accession number of KP403724. The most enzyme production was measured after 72 hours incubation at 20°C in the presence of hazelnut oil as carbon source and yeast extract as nitrogen source and pH 7. The analysis of Taguchi test showed that the most effective factors in enzyme production were carbon source with 54.65% and nitrogen source with 19% of effectiveness. This is the first report of alkaline lipase production by Planomicrobium okeanokoites. This lipase was resistant to low-temperature and 15% saline, so it has wide applications in medical as well as microbial biotechnology.

  13. Semi-continuous in situ magnetic separation for enhanced extracellular protease productionmodeling and experimental validation

    DEFF Research Database (Denmark)

    Cerff, M.; Scholz, A.; Käppler, T.

    2013-01-01

    In modern biotechnology proteases play a major role as detergent ingredients. Especially the production of extracellular protease by Bacillus species facilitates downstream processing because the protease can be directly harvested from the biosuspension. In situ magnetic separation (ISMS...... production, and was used to optimize ISMS steps to obtain the maximum overall protease yield. Biotechnol. Bioeng. 2013; 110: 2161–2172. © 2013 Wiley Periodicals, Inc....

  14. Purification and characterisation of a salt-stable protease from the halophilic archaeon Halogranum rubrum.

    Science.gov (United States)

    Gao, Ruichang; Shi, Tong; Liu, Xiangdong; Zhao, Mengqin; Cui, Henglin; Yuan, Li

    2017-03-01

    Because proteases play an important role in the fermentation of fish sauce, the purification and characterisation of an extracellular protease from the halophilic archaeon Halogranum rubrum was investigated. The molecular mass of the protease was estimated to be approximately 47 kDa based on sodium dodecyl sulfate-polyacrylamide gel electropheresis (SDS-PAGE) and native-PAGE analysis. The optimum conditions for catalytic activity were pH 8.0 and 50°C. The protease showed alkaline stability (pH 7.0-10.0). The protease also exhibited novel catalytic ability over a broad range of salinity (NaCl 0-3 mol L -1 ). Calcium ion enhanced the proteolytic activity of the enzyme. The K m and V max values of the purified protease for casein were calculated to be 4.89 mg mL -1 and 1111.11 U mL -1 , respectively. The protease was strongly inhibited by ethylenediamine tetraacetic acid (EDTA) and phenylmethanesulfonyl fluoride (PMSF). Meanwhile, the protease was stable in the presence of Triton X-100, isopropanol, ethanol or dithio-bis-nitrobenzoic (DTNB), but was inhibited by sodium dodecyl sulfate (SDS), dimethyl sulfoxide (DMSO) or methanol. MALDI -TOF/TOF MS analysis revealed that the protease shared some functional traits with protease produced by Halogranum salarium. Furthermore, it exhibited high hydrolytic activity on silver carp myosin protein. The protease is an alkaline and salt-tolerant enzyme that hydrolyses silver carp myosin with high efficiency. These excellent characteristics make this protease an attractive candidate for industrial use in low-salt fish sauce fermentation. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

  15. Computation of interactive effects and optimization of process parameters for alkaline lipase production by mutant strain of Pseudomonas aeruginosa using response surface methodology

    Directory of Open Access Journals (Sweden)

    Deepali Bisht

    2013-01-01

    Full Text Available Alkaline lipase production by mutant strain of Pseudomonas aeruginosa MTCC 10,055 was optimized in shake flask batch fermentation using response surface methodology. An empirical model was developed through Box-Behnken experimental design to describe the relationship among tested variables (pH, temperature, castor oil, starch and triton-X-100. The second-order quadratic model determined the optimum conditions as castor oil, 1.77 mL.L-1; starch, 15.0 g.L-1; triton-X-100, 0.93 mL.L-1; incubation temperature, 34.12 ºC and pH 8.1 resulting into maximum alkaline lipase production (3142.57 U.mL-1. The quadratic model was in satisfactory adjustment with the experimental data as evidenced by a high coefficient of determination (R² value (0.9987. The RSM facilitated the analysis and interpretation of experimental data to ascertain the optimum conditions of the variables for the process and recognized the contribution of individual variables to assess the response under optimal conditions. Hence Box-Behnken approach could fruitfully be applied for process optimization.

  16. Treatment of PCR products with exonuclease I and heat-labile alkaline phosphatase improves the visibility of combined bisulfite restriction analysis

    International Nuclear Information System (INIS)

    Watanabe, Kousuke; Emoto, Noriko; Sunohara, Mitsuhiro; Kawakami, Masanori; Kage, Hidenori; Nagase, Takahide; Ohishi, Nobuya; Takai, Daiya

    2010-01-01

    Research highlights: → Incubating PCR products at a high temperature causes smears in gel electrophoresis. → Smears interfere with the interpretation of methylation analysis using COBRA. → Treatment with exonuclease I and heat-labile alkaline phosphatase eliminates smears. → The elimination of smears improves the visibility of COBRA. -- Abstract: DNA methylation plays a vital role in the regulation of gene expression. Abnormal promoter hypermethylation is an important mechanism of inactivating tumor suppressor genes in human cancers. Combined bisulfite restriction analysis (COBRA) is a widely used method for identifying the DNA methylation of specific CpG sites. Here, we report that exonuclease I and heat-labile alkaline phosphatase can be used for PCR purification for COBRA, improving the visibility of gel electrophoresis after restriction digestion. This improvement is observed when restriction digestion is performed at a high temperature, such as 60 o C or 65 o C, with BstUI and TaqI, respectively. This simple method can be applied instead of DNA purification using spin columns or phenol/chloroform extraction. It can also be applied to other situations when PCR products are digested by thermophile-derived restriction enzymes, such as PCR restriction fragment length polymorphism (RFLP) analysis.

  17. Evaluation of lignins from side-streams generated in an olive tree pruning-based biorefinery: Bioethanol production and alkaline pulping.

    Science.gov (United States)

    Santos, José I; Fillat, Úrsula; Martín-Sampedro, Raquel; Eugenio, María E; Negro, María J; Ballesteros, Ignacio; Rodríguez, Alejandro; Ibarra, David

    2017-12-01

    In modern lignocellulosic-based biorefineries, carbohydrates can be transformed into biofuels and pulp and paper, whereas lignin is burned to obtain energy. However, a part of lignin could be converted into value-added products including bio-based aromatic chemicals, as well as building blocks for materials. Then, a good knowledge of lignin is necessary to define its valorisation procedure. This study characterized different lignins from side-streams produced from olive tree pruning bioethanol production (lignins collected from steam explosion pretreatment with water or phosphoric acid as catalysts, followed by simultaneous saccharification and fermentation process) and alkaline pulping (lignins recovered from kraft and soda-AQ black liquors). Together with the chemical composition, the structure of lignins was investigated by FTIR, 13 C NMR, and 2D NMR. Bioethanol lignins had clearly distinct characteristics compared to pulping lignins; a certain number of side-chain linkages (mostly alkyl-aryl ether and resinol) accompanied with lower phenolic hydroxyls content. Bioethanol lignins also showed a significant amount of carbohydrates, mainly glucose and protein impurities. By contrast, pulping lignins revealed xylose together with a dramatical reduction of side-chains (some resinol linkages survive) and thereby higher phenol content, indicating rather severe lignin degradation during alkaline pulping processes. All lignins showed a predominance of syringyl units. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Production of bio-oil with low contents of copper and chlorine by fast pyrolysis of alkaline copper quaternary-treated wood in a fluidized bed reactor

    International Nuclear Information System (INIS)

    Koo, Won-Mo; Jung, Su-Hwa; Kim, Joo-Sik

    2014-01-01

    Fast pyrolysis of ACQ (alkaline copper quaternary)-treated wood was carried out in a bench-scale pyrolysis plant equipped with a fluidized bed reactor and char separation system. This study focused on the production of a bio-oil with low copper and chlorine contents, especially by adopting the fractional condensation of bio-oil using water condensers, an impact separator and an electrostatic precipitator. In addition, various analytical tools were applied to investigate the physicochemical properties of the pyrolysis products and the behavior of the preservative during pyrolysis. The bio-oil yield was maximized at 63.7 wt% at a pyrolysis temperature of 411 °C. Highly water-soluble holocellulose-derived components such as acetic acid and hydroxyacetone were mainly collected by the condensers, while lignin-derived components and levoglucosan were mainly observed in the oils collected by the impact separator and electrostatic precipitator. All the bio-oils produced in the experiments were almost free of copper and chlorine. Most copper in ACQ was transferred into the char. - Highlights: • ACQ(alkaline copper quaternary)-treated wood was successfully pyrolyzed in a bench-scale fluidized bed. • Bio-oils separately collected were different in their characteristics. • Bio-oils were free of didecyldimethylammonium chloride. • Bio oils were almost free of copper and chlorine. • The concentration of levoglucosan in a bio-oil was 24–31 wt%

  19. Computation of interactive effects and optimization of process parameters for alkaline lipase production by mutant strain of Pseudomonas aeruginosa using response surface methodology

    Science.gov (United States)

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

    2013-01-01

    Alkaline lipase production by mutant strain of Pseudomonas aeruginosa MTCC 10,055 was optimized in shake flask batch fermentation using response surface methodology. An empirical model was developed through Box-Behnken experimental design to describe the relationship among tested variables (pH, temperature, castor oil, starch and triton-X-100). The second-order quadratic model determined the optimum conditions as castor oil, 1.77 mL.L−1; starch, 15.0 g.L−1; triton-X-100, 0.93 mL.L−1; incubation temperature, 34.12 °C and pH 8.1 resulting into maximum alkaline lipase production (3142.57 U.mL−1). The quadratic model was in satisfactory adjustment with the experimental data as evidenced by a high coefficient of determination (R2) value (0.9987). The RSM facilitated the analysis and interpretation of experimental data to ascertain the optimum conditions of the variables for the process and recognized the contribution of individual variables to assess the response under optimal conditions. Hence Box-Behnken approach could fruitfully be applied for process optimization. PMID:24159311

  20. Fibrin(ogen)olytic activity of bumblebee venom serine protease

    International Nuclear Information System (INIS)

    Qiu Yuling; Choo, Young Moo; Yoon, Hyung Joo; Jia Jingming; Cui Zheng; Wang Dong; Kim, Doh Hoon; Sohn, Hung Dae; Jin, Byung Rae

    2011-01-01

    Bee venom is a rich source of pharmacologically active components; it has been used as an immunotherapy to treat bee venom hypersensitivity, and venom therapy has been applied as an alternative medicine. Here, we present evidence that the serine protease found in bumblebee venom exhibits fibrin(ogen)olytic activity. Compared to honeybee venom, bumblebee venom contains a higher content of serine protease, which is one of its major components. Venom serine proteases from bumblebees did not cross-react with antibodies against the honeybee venom serine protease. We provide functional evidence indicating that bumblebee (Bombus terrestris) venom serine protease (Bt-VSP) acts as a fibrin(ogen)olytic enzyme. Bt-VSP activates prothrombin and directly degrades fibrinogen into fibrin degradation products. However, Bt-VSP is not a plasminogen activator, and its fibrinolytic activity is less than that of plasmin. Taken together, our results define roles for Bt-VSP as a prothrombin activator, a thrombin-like protease, and a plasmin-like protease. These findings offer significant insight into the allergic reaction sequence that is initiated by bee venom serine protease and its potential usefulness as a clinical agent in the field of hemostasis and thrombosis. - Graphical abstract: Display Omitted Highlights: → Bumblebee venom serine protease (Bt-VSP) is a fibrin(ogen)olytic enzyme. → Bt-VSP activates prothrombin. → Bt-VSP directly degrades fibrinogen into fibrin degradation products. → Bt-VSP is a hemostatically active protein that is a potent clinical agent.

  1. Effect of the culture conditions on the production of an extracellular protease by thermophilic Bacillus sp and some properties of the enzymatic activity Efeito das condições de cultivo sobre a produção de proteases extracelulares pelo termofílico Bacillus sp e algumas propriedades da atividade enzimática

    Directory of Open Access Journals (Sweden)

    Camila Rocha da Silva

    2007-06-01

    Full Text Available Protease production by thermophilic Bacillus sp strain SMIA-2 cultivated in liquid cultures containing 1% maltose as a carbon source and supplemented with whey protein (0.1% and corn steep liquor (0.3% reached a maximum at 14 h, with levels of 42 U/mg protein. The microorganism was capable of utilizing a wide range of carbon sources, but protease activity varied according the carbon source. Starch and maltose were the best carbon sources in the present study for protease secretion, while lactose and sucrose were less effective. Increasing maltose concentration in the medium until 1%, improved the growth of the organism and the enzyme activity. Regarding the amounts of corn steep liquor and whey protein in the medium, the concentrations of 0.2% and 0.1% respectively, were considered the most effective for protease secretion by the organism. Studies on the protease characterization revealed that the optimum temperature of this enzyme was 70ºC. Thermostability profile indicated that the enzyme retained 80% of the original activity after 2 h heat treatment at 60ºC. At 70ºC, 70% of the original activity was retained after 15 min heat treatment. The optimum pH of the enzyme was found to be 8.5. After incubation of crude enzyme solution at room temperature for 2 h at pH 6.0-10.0, a decreased of about 15% of its original activity at pH 8.5 was observed. At pH 10.0, the decrease was 24%. In the presence of 1.0 M and 5.0 M NaCl, 76% and 37% of protease activity was retained after 2 h incubating at 45ºC respectively.A produção de proteases pelo termofílico Bacillus sp cepa SMIA-2 cultivado em culturas líquidas contendo maltose (1% e suplementada com proteínas de soro (0,1% e água de maceração de milho (0,3% alcançou o máximo em 14 h, com níveis de 42 U/mg proteína. O microrganismo foi capaz de utilizar várias fontes de carbono, mas a atividade da protease variou com cada fonte. Amido e maltose foram as melhores fontes para a secreção da

  2. Stability and selectivity of alkaline proteases in hydrophilic solvents

    DEFF Research Database (Denmark)

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

    2008-01-01

    , 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......, Carbohydr. Res., 1998, 314, 259. [10]         N.R. Pedersen, R. Wimmer, R. Matthiesen, L.H. Pedersen, A. Gessesse, Tetrahedron: Asymmetry 2003, 14, 667. [11]         L. H. Pedersen, S. Ritthitham and M. Kristensen (2008) in Modern Biocatalysis Eds W. D. Fessner and T. Anthonsen, Wiley-VCH in press...

  3. Alkaline protease from senesced leaves of invasive weed | Gaur ...

    African Journals Online (AJOL)

    step procedure involving ammonium sulfate precipitation and Sephadex G-250 gel permeation chromatography. The Sephadex-G-250 fraction of senesced leaves of Lantana camara showed 28.31 fold with a yield of 6.19%. The enzyme was ...

  4. Characterization of the products attained from a thermal treatment of a mix of zinc-carbon and alkaline batteries.

    Science.gov (United States)

    Kuo, Yi-Ming; Lin, Chitsan; Wang, Jian-Wen; Huang, Kuo-Lin; Tsai, Cheng-Hsien; Wang, Chih-Ta

    2016-01-01

    This study applies a thermal separation process (TSP) to recover Fe, Mn, and Zn from hazardous spent zinc-carbon and alkaline batteries. In the TSP, the batteries were heated together with a reducing additive and the metals in batteries, according to their boiling points and densities, were found to move into three major output materials: slag, ingot (mainly Fe and Mn), and particulate (particularly Zn). The slag well encapsulated the heavy metals of interest and can be recycled for road pavement or building materials. The ingot had high levels of Fe (522,000 mg/kg) and Mn (253,000 mg/kg) and can serve as an additive for stainless steel-making processes. The particulate phase had a Zn level of 694,000 mg/kg which is high enough to be directly sold for refinement. Overall, the TSP effectively recovered valuable metals from the hazardous batteries.

  5. Purification and characterization of an extracellular trypsin-like protease of Fusarium oxysporum var. lini.

    Science.gov (United States)

    Barata, Ricardo Andrade; Andrade, Milton Hercules Guerra; Rodrigues, Roberta Dias; Castro, Ieso Miranda

    2002-01-01

    An alkaline serineprotease, capable of hydrolyzing Nalpha-benzoyl- dl arginine p-nitroanilide, was secreted by Fusarium oxysporum var. lini grown in the presence of gelatin as the sole nitrogen and carbon source. The protease was purified 65-fold to electrophoretic homogenity from the culture supernatant in a three-step procedure comprising QSepharose chromatography, affinity chromatography, and FPLC on a MonoQ column. SDS-PAGE analysis of the purified protein indicated an estimated molecular mass of 41 kDa. The protease had optimum activity at a reaction temperature of 45 degrees C and showed a rapid decrease of activity at 48 degrees C. The optimum pH was around 8.0. Characterization of the protease showed that Ca2+ and Mg2+ cations increased the activity, which was not inhibited by EDTA or 1,10-phenanthroline. The enzyme activity on Nalpha-benzoyl-DL arginine p-nitroanilide was inhibited by 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride, p-aminobenzamidine dihydrochloride, aprotinin, 3-4 dichloroisocoumarin, and N-tosyl-L-lysine chloromethyl ketone. The enzyme is also inhibited by substrate concentrations higher than 2.5 x 10(-4)M. The protease had a Michaelis-Menten constant of 0.16 mM and a V(max) of 0.60 mumol released product.min(-1).mg(-1) enzyme when assayed in a non-inhibiting substrate concentration. The activity on Nalpha-benzoyl- dl arginine p-nitroanilide was competitively inhibited by p-aminobenzamidine dihydrochoride. A K(i) value of 0.04 mM was obtained.

  6. Extracellular protease produced by Bacillus subtilis isolated from ...

    African Journals Online (AJOL)

    In a study to evaluate the microbiological safety of some paracetamol oral solutions sold in some Nigerian drug stores, 40.0% of the samples examined was contaminated with protease-producing Bacillus subtilis. The production of extracellular protease was induced by casein in the minimal medium and was found to be the ...

  7. Isolation of protease producing novel Bacillus cereus and detection ...

    African Journals Online (AJOL)

    user

    2011-02-14

    Feb 14, 2011 ... The highest protease activity was determined at 30°C temperature and 6.4 pH conditions and after the 18th hour, it decreased evidently. Key words: Protease, production, optimization, Bacillus sp. INTRODUCTION. Enzymes have been produced in large industrial scale for several decades (Falch, 1991).

  8. Menstrual blood loss measurement: validation of the alkaline hematin technique for feminine hygiene products containing superabsorbent polymers.

    Science.gov (United States)

    Magnay, Julia L; Nevatte, Tracy M; Dhingra, Vandana; O'Brien, Shaughn

    2010-12-01

    To validate the alkaline hematin technique for measurement of menstrual blood loss using ultra-thin sanitary towels that contain superabsorbent polymer granules as the absorptive agent. Laboratory study using simulated menstrual fluid (SMF) and Always Ultra Normal, Long, and Night "with wings" sanitary towels. Keele Menstrual Disorders Laboratory. None. None. Recovery of blood, linearity, and interassay variation over a range of SMF volumes applied to towels. Because of the variable percentage of blood in menstrual fluid, blood recovery was assessed from SMF constituted as 10%, 25%, 50%, and 100% blood. The lower limit of reliable detection and the effect of storing soiled towels for up to 4 weeks at 15°C-20°C, 4°C, and -20°C before analysis were determined. Ninety percent recovery was reproducibly achieved up to 30 mL applied volume at all tested SMF compositions, except at low volume or high dilution equivalent to sanitary towels that contain superabsorbent polymers. Copyright © 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  9. 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.

  10. Transesterification of rapeseed oil for biodiesel production in trickle-bed reactors packed with heterogeneous Ca/Al composite oxide-based alkaline catalyst.

    Science.gov (United States)

    Meng, Yong-Lu; Tian, Song-Jiang; Li, Shu-Fen; Wang, Bo-Yang; Zhang, Min-Hua

    2013-05-01

    A conventional trickle bed reactor and its modified type both packed with Ca/Al composite oxide-based alkaline catalysts were studied for biodiesel production by transesterification of rapeseed oil and methanol. The effects of the methanol usage and oil flow rate on the FAME yield were investigated under the normal pressure and methanol boiling state. The oil flow rate had a significant effect on the FAME yield for the both reactors. The modified trickle bed reactor kept over 94.5% FAME yield under 0.6 mL/min oil flow rate and 91 mL catalyst bed volume, showing a much higher conversion and operational stability than the conventional type. With the modified trickle bed reactor, both transesterification and methanol separation could be performed simultaneously, and glycerin and methyl esters were separated additionally by gravity separation. Copyright © 2013 Elsevier Ltd. All rights reserved.

  11. Alkaline pH Is a signal for optimal production and secretion of the heat labile toxin, LT in enterotoxigenic Escherichia coli (ETEC.

    Directory of Open Access Journals (Sweden)

    Lucia Gonzales

    Full Text Available Enterotoxigenic Escherichia coli (ETEC cause secretory diarrhea in children and travelers to endemic areas. ETEC spreads through the fecal-oral route. After ingestion, ETEC passes through the stomach and duodenum before it colonizes the lower part of the small intestine, exposing bacteria to a wide range of pH and environmental conditions. This study aimed to determine the impact of external pH and activity of the Cyclic AMP receptor protein (CRP on the regulation of production and secretion of heat labile (LT enterotoxin. ETEC strain E2863wt and its isogenic mutant E2863ΔCRP were grown in LBK media buffered to pH 5, 7 and 9. GM1 ELISA, cDNA and cAMP analyses were carried out on bacterial pellet and supernatant samples derived from 3 and 5 hours growth and from overnight cultures. We confirm that CRP is a repressor of LT transcription and production as has been shown before but we show for the first time that CRP is a positive regulator of LT secretion both in vitro and in vivo. LT secretion increased at neutral to alkaline pH compared to acidic pH 5 where secretion was completely inhibited. At pH 9 secretion of LT was optimal resulting in 600 percent increase of secreted LT compared to unbuffered LBK media. This effect was not due to membrane leakage since the bacteria were viable at pH 9. The results indicate that the transition to the alkaline duodenum and/or exposure to high pH close to the epithelium as well as activation of the global transcription factor CRP are signals that induce secretion of the LT toxin in ETEC.

  12. Chitosan nanofiber scaffold improves bone healing via stimulating trabecular bone production due to upregulation of the Runx2/osteocalcin/alkaline phosphatase signaling pathway

    Science.gov (United States)

    Ho, Ming-Hua; Yao, Chih-Jung; Liao, Mei-Hsiu; Lin, Pei-I; Liu, Shing-Hwa; Chen, Ruei-Ming

    2015-01-01

    Osteoblasts play critical roles in bone formation. Our previous study showed that chitosan nanofibers can stimulate osteoblast proliferation and maturation. This translational study used an animal model of bone defects to evaluate the effects of chitosan nanofiber scaffolds on bone healing and the possible mechanisms. In this study, we produced uniform chitosan nanofibers with fiber diameters of approximately 200 nm. A bone defect was surgically created in the proximal femurs of male C57LB/6 mice, and then the left femur was implanted with chitosan nanofiber scaffolds for 21 days and compared with the right femur, which served as a control. Histological analyses revealed that implantation of chitosan nanofiber scaffolds did not lead to hepatotoxicity or nephrotoxicity. Instead, imaging analyses by X-ray transmission and microcomputed tomography showed that implantation of chitosan nanofiber scaffolds improved bone healing compared with the control group. In parallel, microcomputed tomography and bone histomorphometric assays further demonstrated augmentation of the production of new trabecular bone in the chitosan nanofiber-treated group. Furthermore, implantation of chitosan nanofiber scaffolds led to a significant increase in the trabecular bone thickness but a reduction in the trabecular parameter factor. As to the mechanisms, analysis by confocal microscopy showed that implantation of chitosan nanofiber scaffolds increased levels of Runt-related transcription factor 2 (Runx2), a key transcription factor that regulates osteogenesis, in the bone defect sites. Successively, amounts of alkaline phosphatase and osteocalcin, two typical biomarkers that can simulate bone maturation, were augmented following implantation of chitosan nanofiber scaffolds. Taken together, this translational study showed a beneficial effect of chitosan nanofiber scaffolds on bone healing through stimulating trabecular bone production due to upregulation of Runx2-mediated alkaline

  13. 2-D zymographic analysis of Broccoli (Brassica oleracea L. var. Italica) florets proteases: follow up of cysteine protease isotypes in the course of post-harvest senescence.

    Science.gov (United States)

    Rossano, Rocco; Larocca, Marilena; Riccio, Paolo

    2011-09-01

    Zymographic analysis of Broccoli florets (Brassica oleracea L. var. Italica) revealed the presence of acidic metallo-proteases, serine proteases and cysteine proteases. Under conditions which were denaturing for the other proteases, the study was restricted to cysteine proteases. 2-D zymography, a technique that combines IEF and zymography was used to show the presence of 11 different cysteine protease spots with molecular mass of 44 and 47-48kDa and pIs ranging between 4.1 and 4.7. pI differences could be ascribed to different degrees of phosphorylation that partly disappeared in the presence of alkaline phosphatase. Post-harvest senescence of Broccoli florets was characterized by decrease in protein and chlorophyll contents and increase of protease activity. In particular, as determined by 2-D zymography, the presence of cysteine protease clearly increased during senescence, a finding that may represent a useful tool for the control of the aging process. Copyright © 2011 Elsevier GmbH. All rights reserved.

  14. Nota Científica: utilização da pectina, proteínas do soro de queijo e água de maceração de milho para a produção de proteases por Bacillus sp. termofílico Scientific Note: use of pectin, whey protein and corn steep liquor for the production of protease by thermophilic Bacillus sp.

    Directory of Open Access Journals (Sweden)

    Silvania Alves Ladeira

    2012-03-01

    Full Text Available As enzimas proteolíticas termoestáveis produzidas por microrganismos do gênero Bacillus possuem grande importância comercial, sendo sua aplicação predominante (35% na indústria de detergentes. Neste trabalho, foi avaliada a produção de proteases pelo termofílico Bacillus sp. SMIA-2, utilizando-se substratos de baixo custo. A fim de verificar a utilidade da protease para aplicações industriais, a estabilidade e a atividade da enzima a diferentes valores de pH e temperatura foram também estudadas. A atividade da protease secretada por Bacillus sp. SMIA-2 em culturas submersas contendo 0,5% (m/v de pectina de maçã, 0,1% (m/v de proteínas do soro e 0,3% (m/v de água de maceração de milho foi máxima após 24 h de incubação da cultura, com níveis de 54,3 U.mg-1 Proteína. A redução na concentração da pectina para 0,3% (m/v e o aumento nos níveis das proteínas do soro para 0,3% (m/v no meio de cultura aumentaram a produção da protease, que alcançou sua máxima atividade em 30 h, com níveis de 72,2 U.mg-1 Proteína. Estudos sobre a protease revelaram que as suas características mais importantes foram a alta temperatura ótima para atividade da enzima (70 °C e a alta estabilidade em uma grande faixa de pH. A protease reteve em torno de 80% de sua atividade original quando incubada à temperatura ambiente por 2 h na faixa de pH entre 6,0 e 12,0. Essas propriedades constituem importantes vantagens para um possível uso da enzima em indústrias de detergentes.The thermostable proteolytic enzymes produced by the genus Bacillus are commercially very important, being predominantly applied (35% in detergents. In this work the production of proteases by a thermophilic Bacillus sp. SMIA-2 using low-cost substrates was evaluated. In order to assess the use of the protease for industrial use, the stability of the enzyme activity at different pH values and temperatures was also studied. The protease activity secreted by Bacillus sp

  15. Nucleic Acid Aptamers Against Proteases

    DEFF Research Database (Denmark)

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

    2011-01-01

    , 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......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...... small molecule protease inhibitors of sufficient specificity has proved a daunting task. Aptamers seem to represent a promising alternative. In our review, we concentrate on biochemical mechanisms of aptamer selection, proteinaptamer recognition, protease inhibition, and advantages of aptamers...

  16. Cloning and enhancing production of a detergent- and organic-solvent-resistant nattokinase from Bacillus subtilis VTCC-DVN-12-01 by using an eight-protease-gene-deficient Bacillus subtilis WB800.

    Science.gov (United States)

    Nguyen, Thao Thi; Quyen, Thi Dinh; Le, Hoang Thanh

    2013-09-10

    Nattokinases/Subtilisins (EC 3.4.21.62) belong to the second large family of serine proteases, which gain significant attention and play important role in many biotechnology processes. Thus, a number of nattokinases/subtilisins from various Bacillus species, especially from B. subtilis strains, extensively have been investigated to understand their biochemical and physical properties as well as to improve the production for industrial application. The purpose of this study was to clone a nattokinase gene from Bacillus subtilis strain VTCC-DVN-12-01, enhance its production in B. subtilis WB800, which is deficient in eight extracellular proteases and characterize its physicochemical properties for potential application in organic synthesis and detergent production. A gene coding for the nattokinase (Nk) from B. subtilis strain VTCC-DVN-12-01 consisted of an ORF of 1146 nucleotides, encoding a pre-pro-protein enzyme (30-aa pre-signal peptide, 76-aa pro-peptide and 275-aa mature protein with a predicted molecular mass of 27.7 kDa and pI 6.6). The nattokinase showed 98-99% identity with other nattokinases/subtilisins from B. subtilis strains in GenBank. Nk was expressed in B. subtilis WB800 under the control of acoA promoter at a high level of 600 mg protein per liter culture medium which is highest yield of proteins expressed in any extracellular-protease-deficient B. subtilis system till date. Nk was purified to homogeneity with 3.25 fold purification, a specific activity of 12.7 U/mg, and a recovery of 54.17%. The purified Nk was identified by MALDI-TOF mass spectrometry through three peptides, which showed 100% identity to corresponding peptides of the B. subtilis nattokinase (CAC41625). An optimal activity for Nk was observed at 65 °C and pH 9. The nattokinase was stable at temperature up to 50 °C and in pH range of 5-11 and retained more than 85% of its initial activity after incubation for 1 h. Mg2+ activated Nk up to 162% of its activity. The addition of

  17. 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. Copyright © 2016 Elsevier Ltd. All rights reserved.

  18. Metal complexation in near field conditions of nuclear waste repository - stability constant of copper complexation with cellulose degradation products, in alkaline conditions

    International Nuclear Information System (INIS)

    Guede, Kipre Bertin

    2005-11-01

    Copper is a stable element and spent fuel component which constitutes the radioactive waste. The reaction of Copper with cellulose degradation products in alkaline conditions was performed to mimic what occurs in near field conditions of nuclear waste repository. From the characteristics of Cu (II), this thesis aims at inferring the behaviour of radionuclides vis a vis the degradation products of cellulose. The contribution of the present work is therefore the assessment of the stability of the major cellulose degradation product, its affinity for Copper and the extent of the complexation function 13 between Cu (II) and the organic moieties. The formation of cellulose degradation products was followed by measurement of p11, Conductivity, Angle of rotation, relative abundance of aliphatics and aromatics (E4/E6 ) aid by UV-visible spectroscopy. The TOC was determined using the Walkley and Black titration after respectively 31 weeks and 13 weeks of degradation for the reaction mixtures T and A, N. The stability of the major degradation products gave the following figures: ISA(A): - 13 43.39 <ΔG -10639.88 ISA(N): - Ii 436.45<ΔG< -9103.6. The study of the characteristics of Gluconic Acid, as a model compound, was carried out in an attempt to give a general picture of the roper ties of cellulose degradation products. The Complexation between Cu (II) and the organic ligand (Cellulose degradation products) was performed using UV-visible spectroscopy and Ion Distribution technique. The Log B value obtained from the complexation studies at 336 nm for 1 = 0. I Ni NaClO4 and I = 0.01 M NaClO4, falls within a range of 3.48 to 3.74 for the standard reference material (Gluconic Acid), and within I .87 to 2.3 I, and I .6 to 2.01, respectively for the degradation Products ISA (A) and ISA(N). The ion distribution studies showed that: • In (he absence of the degradation product ISA and at pH = 3.68. 56. 17 % of Cu (II) was bound to the resin. • In the presence of ISA and at 2

  19. Multifunctional Mitochondrial AAA Proteases.

    Science.gov (United States)

    Glynn, Steven E

    2017-01-01

    Mitochondria perform numerous functions necessary for the survival of eukaryotic cells. These activities are coordinated by a diverse complement of proteins encoded in both the nuclear and mitochondrial genomes that must be properly organized and maintained. Misregulation of mitochondrial proteostasis impairs organellar function and can result in the development of severe human diseases. ATP-driven AAA+ proteins play crucial roles in preserving mitochondrial activity by removing and remodeling protein molecules in accordance with the needs of the cell. Two mitochondrial AAA proteases, i-AAA and m-AAA, are anchored to either face of the mitochondrial inner membrane, where they engage and process an array of substrates to impact protein biogenesis, quality control, and the regulation of key metabolic pathways. The functionality of these proteases is extended through multiple substrate-dependent modes of action, including complete degradation, partial processing, or dislocation from the membrane without proteolysis. This review discusses recent advances made toward elucidating the mechanisms of substrate recognition, handling, and degradation that allow these versatile proteases to control diverse activities in this multifunctional organelle.

  20. Diversity of both the cultivable protease-producing bacteria and bacterial extracellular proteases in the coastal sediments of King George Island, Antarctica.

    Directory of Open Access Journals (Sweden)

    Ming-Yang Zhou

    Full Text Available Protease-producing bacteria play a vital role in degrading sedimentary organic nitrogen. However, the diversity of these bacteria and their extracellular proteases in most regions remain unknown. In this paper, the diversity of the cultivable protease-producing bacteria and of bacterial extracellular proteases in the sediments of Maxwell Bay, King George Island, Antarctica was investigated. The cultivable protease-producing bacteria reached 10(5 cells/g in all 8 sediment samples. The cultivated protease-producing bacteria were mainly affiliated with the phyla Actinobacteria, Firmicutes, Bacteroidetes, and Proteobacteria, and the predominant genera were Bacillus (22.9%, Flavobacterium (21.0% and Lacinutrix (16.2%. Among these strains, Pseudoalteromonas and Flavobacteria showed relatively high protease production. Inhibitor analysis showed that nearly all the extracellular proteases from the bacteria were serine proteases or metalloproteases. These results begin to address the diversity of protease-producing bacteria and bacterial extracellular proteases in the sediments of the Antarctic Sea.

  1. Thermo-alkaline pretreatment of waste activated sludge at low-temperatures: effects on sludge disintegration, methane production, and methanogen community structure.

    Science.gov (United States)

    Kim, Jaai; Yu, Youngseob; Lee, Changsoo

    2013-09-01

    Low-temperature thermo-alkaline pretreatment of waste activated sludge (WAS) was studied, within the region of 0-0.2 M NaOH and 60-90°C, for the effects of NaOH concentration and temperature on sludge degradability in anaerobic digestion (AD). Significant disintegration of sludge solids (up to 75.6%) and an increase in methane production (up to 70.6%) were observed in the pretreatment trials. Two quadratic models were successfully generated by response surface analysis (R(2)>0.9, pdisintegration (SD) and methane production (MP) respond to changes in the pretreatment conditions. The maximum responses of SD (77.8%) and MP (73.9% increase over the control) were shown at [0.16 M NaOH, 90°C] and [0.10 M NaOH, 73.7°C], respectively. NaOH addition showed a significant influence on the evolution of methanogen community structure during AD, whereas temperature did not. Aceticlastic Methanosaeta and Methanosarcina speceies were likely the major methanogens. Copyright © 2013 Elsevier Ltd. All rights reserved.

  2. Biodiesel Production from Kapok (Ceiba pentandra Seed Oil using Naturally Alkaline Catalyst as an Effort of Green Energy and Technology

    Directory of Open Access Journals (Sweden)

    N.A. Handayani

    2013-10-01

    Full Text Available Nowadays, energy that used to serve all the needs of community, mainly generated from fossil (conventional energy. Terrace in energy consumption is not balanced with adequate fossil fuel reserves and will be totally depleted in the near future. Indonesian Government through a Presidential Decree No. 5 year 2006 mandates an increased capacity in renewable energy production from 5 percent to 15 percent in 2025. C. pentandra seed oil has feasibility as a sustainable biodiesel feedstock in Indonesia. The aim of this paper was to investigate biodiesel production from ceiba petandra seed oil using naturally potassium hydroxide catalyst. Research designs are based on factorial design with 2 levels and 3 independent variables (temperature, reaction time and molar ratio of methanol to oil. According to data calculation, the most influential single variable is molar ratio of methanol to oil. Characterization of biodiesel products meet all the qualifications standardized by SNI 04-7182-2006. Keywords: biodiesel, kapok seed oil, c. pentandra, green technology

  3. Effect of Light/Dark Regimens on Hydrogen Production by Tetraselmis subcordiformis Coupled with an Alkaline Fuel Cell System.

    Science.gov (United States)

    Guo, Zhen; Li, Ying; Guo, Haiyan

    2017-12-01

    To improve the photoproduction of hydrogen (H 2 ) by a green algae-based system, the effect of light/dark regimens on H 2 photoproduction regulated by carbonyl cyanide m-chlorophenylhydrazone (CCCP) was investigated. A fuel cell was integrated into a photobioreactor to allow online monitoring of the H 2 evolution rate and decrease potential H 2 feedback inhibition by consuming the generated H 2 in situ. During the first 15 h of H 2 evolution, the system was subjected to dark treatment after initial light illumination (L/D = 6/9 h, 9/6 h, and 12/3 h). After the dark period, all systems were again exposed to light illumination until H 2 evolution stopped. Two peaks were observed in the H 2 evolution rate under all three light/dark regimens. Additionally, a high H 2 yield of 126 ± 10 mL L -1 was achieved using a light/dark regimen of L 9 h/D 6 h/L until H 2 production ceased, which was 1.6 times higher than that obtained under continuous illumination. H 2 production was accompanied by some physiological and morphological changes in the cells. The results indicated that light/dark regimens improved the duration and yield of H 2 photoproduction by the CCCP-regulated process of Tetraselmis subcordiformis.

  4. Photocatalytic and photoelectrocatalytic degradation of the drug omeprazole on nanocrystalline titania films in alkaline media: Effect of applied electrical bias on degradation and transformation products

    Energy Technology Data Exchange (ETDEWEB)

    Tantis, Iosif [Department of Chemical Engineering, University of Patras, Caratheodory 1, University Campus, GR-26504 Patras (Greece); Bousiakou, Leda [Department of Physics and Astronomy, King Saud University, Riyadh (Saudi Arabia); Department of Automation Engineering, Technological Educational Institute of Pireaus, GR-12244 Athens (Greece); Frontistis, Zacharias; Mantzavinos, Dionissios [Department of Chemical Engineering, University of Patras, Caratheodory 1, University Campus, GR-26504 Patras (Greece); Konstantinou, Ioannis; Antonopoulou, Maria [Department of Environmental and Natural Resources Management, University of Patras, GR-30100 Agrinio (Greece); Karikas, George-Albert [Department of Medical Laboratories Technology, Technological Educational Institute of Athens, 12210 Athens (Greece); Lianos, Panagiotis, E-mail: lianos@upatras.gr [Department of Chemical Engineering, University of Patras, Caratheodory 1, University Campus, GR-26504 Patras (Greece); FORTH/ICE-HT, P.O. Box 1414, GR-26504 Patras (Greece)

    2015-08-30

    Highlights: • Photocatalytic and photoelectrocatalytic degradation of the proton pump omeprazole. • Improvement of photocatalysis rate by applying a moderate forward bias. • Highlighting of the advantages of photoelectrocatalysis in a straightforward manner. • HPLC and HR-LC–MS analysis of transformation products. - Abstract: Photocatalytic and photoelectrocatalytic degradation of the drug omeprazole has been studied in the presence of nanocrystalline titania films supported on glass slides or transparent FTO electrodes in alkaline environment. Its photocatalytic degradation rate was assessed by its UV absorbance and by HPLC, while its transformation products were analyzed by HR-LC–MS. Based on UV absorbance, omeprazole can be photocatalytically degraded at an average rate of 6.7 × 10{sup −4} min{sup −1} under low intensity UVA irradiation of 1.5 mW cm{sup −2} in the presence of a nanoparticulate titania film. This corresponds to degradation of 1.4 mg of omeprazole per gram of the photocatalyst per liter of solution per hour. The photodegradation rate can be accelerated in a photoelectrochemical cell by applying a forward bias. In this case, the maximum rate reached under the present conditions was 11.6 × 10{sup −4} min{sup −1} by applying a forward bias of +0.6 V vs. Ag/AgCl. Four major transformation products were successfully identified and their profiles were followed by HR-LC–MS. The major degradation path includes the scission of the sulfoxide bridge into the corresponding pyridine and benzimidazole ring derivates and this is accompanied by the release of sulfate anions in the reaction mixture.

  5. Hydrogen and syngas production by catalytic gasification of algal biomass (Cladophora glomerata L.) using alkali and alkaline-earth metals compounds.

    Science.gov (United States)

    Ebadi, Abdol Ghaffar; Hisoriev, Hikmat; Zarnegar, Mohammad; Ahmadi, Hamed

    2018-01-02

    The steam gasification of algal biomass (Cladophora glomerata L.) in presence of alkali and alkaline-earth metal compounds catalysts was studied to enhance the yield of syngas and reduce its tar content through cracking and reforming of condensable fractions. The commercial catalysts used include NaOH, KHCO 3 , Na 3 PO 4 and MgO. The gasification runs carried out with a research scale, biomass gasification unit, show that the NaOH has a strong potential for production of hydrogen, along with the added advantages of char converting and tar destruction, allowing enhancement of produced syngas caloric value. When the temperature increased from 700°C to 900°C, the tar content in the gas sharply decreased, while the hydrogen yield increased. Increasing steam/biomass ratio significantly increased hydrogen yield and tar destruction; however, the particle size in the range of 0.5-2.5 mm played a minor role in the process.

  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. Production of Cellulases, Xylanase, Pectinase, alpha-amylase and Protease Enzymes Cocktail by Bacillus spp. and Their Mixed Cultures with Candida tropicalis and Rhodotorula glutinis under Solid State Fermentation

    International Nuclear Information System (INIS)

    El-Batal, A.I.; Abo-State, M.A.

    2006-01-01

    A group of twelve locally isolated Bacillus species, B.megaterium (MAI and MA II), B.licheniformis (MLI and ML II); B. circulans, B. stearothermophilis, B.cereus, B.sphaericus, B. pumilus, B. laterosporus, B. coagulans and B. pantothenticus, were examined for the production of cellulases, xylanase, pectinase, alpha-amylase and protease enzymes cocktail on wheat bran under solid state fermentation (SSF). All species were found to be potent hydrolyzing enzymes producers and the superior producing species were B. megaterium MAI and B. licheniformis. On the other hand, both of them still produced highest enzyme titres when mixed with Candida tropicalis or Rhodotorula glutinis, yeast strains. The two superior bacterial strains produced the highest enzymatic activities when coculturing with C. tropicalis compared with coculturing with R. glutinis only or with both C. tropicalis and R. glutinis in combination. The inferior activities of cocultures (B. megaterinm MAI and R. glutinis) were enhanced in carboxymethyl cellulase (CMCase), filter paper cellulase (FPase), avecilase, xylanase, pectinase, -amylase and protease by gamma irradiation at dose 1.0 kGy with percent increase 8 %, 20 %, 10 %, 4 %, 31 %, 22 % and 34 %, respectively as compared with un-irradiated cocultures

  8. Two and three way spectrophotometric-assisted multivariate determination of linezolid in the presence of its alkaline and oxidative degradation products and application to pharmaceutical formulation

    Science.gov (United States)

    Hegazy, Maha Abd El-Monem; Eissa, Maya Shaaban; Abd El-Sattar, Osama Ibrahim; Abd El-Kawy, Mohammad

    2014-07-01

    Linezolid (LIN) is determined in the presence of its alkaline (ALK) and oxidative (OXD) degradation products without preliminary separation based on ultraviolet spectrophotometry using two-way chemometric methods; principal component regression (PCR) and partial least-squares (PLS), and three-way chemometric methods; parallel factor analysis (PARAFAC) and multi-way partial least squares (N-PLS). A training set of mixtures containing LIN, ALK and OXD; was prepared in the concentration ranges of 12-18, 2.4-3.6 and 1.2-1.8 μg mL-1, respectively according to a multilevel multifactor experimental design. The multivariate calibrations were obtained by measuring the zero-order absorbance from 220 to 320 nm using the training set. The validation of the multivariate methods was realized by analyzing their synthetic mixtures. The capabilities of the chemometric analysis methods for the analysis of real samples were evaluated by determination of LIN in its pharmaceutical preparation with satisfactory results. The accuracy of the methods, evaluated through the root mean square error of prediction (RMSEP), was 0.058, 0.026, 0.101 and 0.026 for LIN using PCR, PLS, PARAFAC and N-PLS, respectively. Protolytic equilibria of LIN and its degradation products were evaluated using the corresponding absorption spectra-pH data obtained with PARAFAC. The obtained pKa values of LIN, ALK and OXD are 5.70, 8.90 and 6.15, respectively. The results obtained were statistically compared to that of a reported HPLC method, and there was no significant difference between the proposed methods and the reported method regarding both accuracy and precision.

  9. Processing Methods of Alkaline Hydrolysate from Rice Husk

    Directory of Open Access Journals (Sweden)

    Olga D. Arefieva

    2017-07-01

    Full Text Available This paper devoted to finding processing methods of alkaline hydrolysate produced from rice husk pre-extraction, and discusses alkaline hydrolysate processing schemed and disengagement of some products: amorphous silica of various quality, alkaline lignin, and water and alkaline extraction polysaccharides. Silica samples were characterized: crude (air-dried, burnt (no preliminary water treatment, washed in distilled water, and washed in distilled water and burnt. Waste water parameters upon the extraction of solids from alkaline hydrolysate dropped a few dozens or thousand times depending on the applied processing method. Color decreased a few thousand times, turbidity was virtually eliminated, chemical oxygen demanded about 20–136 times; polyphenols content might decrease 50% or be virtually eliminated. The most prospective scheme obtained the two following solid products from rice husk alkaline hydrolysate: amorphous silica and alkaline extraction polysaccharide. Chemical oxygen demand of the remaining waste water decreased about 140 times compared to the silica-free solution.

  10. Effect of Seed Proteinaceous Inhibitors on Digestive Protease Activity of Tomato Leaf Miner Tuta absoluta (Meyrick (Lep.: Gelechiidae

    Directory of Open Access Journals (Sweden)

    M. University of Tehran

    2017-01-01

    Full Text Available Introduction: Tomato leaf miner Tuta absoluta Meyrick (Lep.: Gelechiidae is one of the most important pest of tomato around the world which have been introduced to Iran in recent years. Pesticides can usually reduce the damage caused by the pest, however the extensive use of pesticides substantially increase production costs and lead to development of insecticide resistance. Thus, pest resistance and also adverse effects of pesticides on the human health and the environment make search for alternative control method/s inevitable. The aim of this study was to examine the effect of proteinaceous extracts of Datura (Datura stramonium L., Bean (Phaseolus vulgaris L., Wild Oat (Avena fatua L., Broad bean (Vicia faba L., Amaranth (Amaranthus retroflexus L., Mung bean (Vigna radiate L., Pea (Pisum sativum L., Canola (Brassica napus L., Triticale (Triticosecale wittmack and Sivand, Aflak and Alborz wheat (Triticum aestivum L. cultivars seeds on protease activity of tomato leaf miner. Also in this study optimum pH for protease activity and effects of pH on inhibitory activity of extracts were determined. Materials and Methods: For this work fourth instar larvae of Tuta absoluta was used for enzyme extraction. Plant proteinaceous extracts were obtained using 0.1 M NaCl. For protease activity assay 10 μl enzyme extract and 50 μl substrate solution (Azocasein 2% were mixed with 40 μl of the 20 mM Glycine-NaOH buffer at pH 10.0 After 60 min incubation, 100 μl of 30% trichloroacetic acid (TCA was added to the reaction mixture, and kept at 4 °C for 30 min, followed by centrifugation at 15,000gfor 15 min to precipitate non-hydrolysis substrate. 100 μl of 1 M NaOH was added to 100μl supernatant and the absorbance at 405 nm was measured. To determine the effect of the seed proteinaceous extracts on the protease activities, enzymes were pre-incubated with each seed extracts for 30 min at 35 °C followed by determination of the residual enzyme activity as

  11. By-products of the serpentinization process on the Oman ophiolite : chemical and isotopic composition of carbonate deposits in alkaline springs, and associated secondary phases

    Science.gov (United States)

    Sissmann, O.; Martinez, I.; Deville, E.; Beaumont, V.; Pillot, D.; Prinzhofer, A.; Vacquand, C.; Chaduteau, C.; Agrinier, P.; Guyot, F. J.

    2014-12-01

    The isotopic compositions (d13C, d18O) of natural carbonates produced by the alteration of basic and ultrabasic rocks on the Oman ophiolite have been measured in order to better understand their formation mechanisms. Fossil carbonates developed on altered peridotitic samples, mostly found in fractures, and contemporary carbonates were studied. The samples bear a large range of d13C. Those collected in veins are magnesian (magnesite, dolomite) and have a carbon signature reflecting mixing of processes and important fractionation (-11‰ to 8‰). Their association with talc and lizardite suggests they are by-products of a serpentinization process, that must have occurred as a carbon-rich fluid was circulating at depth. On the other hand, the carbonates are mostly calcic when formed in alkaline springs, most of which are located in the vicinity of lithological discontinuities such as the peridotite-gabbro contact (Moho). Aragonite forms a few meters below the surface of the ponds in Mg-poor water, and is systematically associated with brucite (Mg(OH)2). This suggests most of the Mg dissolved at depth has reprecipitated during the fluid's ascension through fractures or faults as carbonates and serpentine. Further up, on the surface waters of the ponds (depleted in Mg and D.I.C.), thin calcite films precipitate and reach extremely negative d13C values (-28‰), which could reflect either a biological carbon source, or kinetic fractionation from pumping atmospheric CO2. Their formation represent an efficient and natural process for carbon dioxide mineral sequestration. The d18O signature from all samples confirm the minerals crystallized from a low-temperature fluid. The hyperalkaline conditions (pH between 11 and 12) allowing for these fast precipitation kinetics are generated by the serpentinization process occurring at depth, as indicated by the measured associated H2-rich gas flows (over 50%) seeping out to the surface.

  12. High temperature and pressure alkaline electrolysis

    DEFF Research Database (Denmark)

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

    2013-01-01

    Alkaline electrolyzers have proven to operate reliable for decades on a large scale, but in order to become commercially attractive and compete against conventional technologies for hydrogen production, the production and investment costs have to be reduced. This may occur by increasing the opera......Alkaline electrolyzers have proven to operate reliable for decades on a large scale, but in order to become commercially attractive and compete against conventional technologies for hydrogen production, the production and investment costs have to be reduced. This may occur by increasing...

  13. 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.

  14. Distinct properties of proteases and nucleases in the gut, salivary gland and saliva of southern green stink bug, Nezara viridula

    Science.gov (United States)

    Lomate, Purushottam R.; Bonning, Bryony C.

    2016-01-01

    Stink bugs negatively impact numerous plant species of agricultural and horticultural importance. While efforts to develop effective control measures are underway, the unique digestive physiology of these pests presents a significant hurdle for either protein- or nucleotide-based management options. Here we report the comparative biochemical and proteomic characterization of proteases and nucleases from the gut, salivary gland and saliva of the southern green stink bug, Nezara viridula. The pH optimum for protease activity was acidic (5 to 6) in the gut with the primary proteases being cysteine proteases, and alkaline (8 to 9) in the saliva and salivary gland with the primary proteases being serine proteases. The serine proteases in saliva differ biochemically from trypsin and chymotrypsin, and the cathepsins in the gut and saliva showed distinct properties in inhibitor assays. Nuclease activity (DNase, RNase, dsRNase) was concentrated in the salivary gland and saliva with negligible activity in the gut. The most abundant proteins of the gut (530) and salivary gland (631) identified by proteomic analysis included four gut proteases along with eight proteases and one nuclease from the salivary gland. Understanding of N. viridula digestive physiology will facilitate the design of new strategies for management of this significant pest. PMID:27282882

  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...... in the effect of proteases on CD4- and CD8-positive cells. To determine the effect of proteases on interleukin-2 (IL-2)-induced cell proliferation, the proteases and IL-2 were added to the IL-2-dependent CTLL-2 cell line. AP and ELA inhibited the proliferation of these cells. When IL-2 was added in excess......, 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...

  16. Uranium in alkaline rocks

    International Nuclear Information System (INIS)

    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

  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. Co-evolution of insect proteases and plant protease inhibitors.

    Science.gov (United States)

    Jongsma, Maarten A; Beekwilder, Jules

    2011-08-01

    Plants are at the basis of the food chain, but there is no such thing as a "free lunch" for herbivores. To promote reproductive success, plants evolved multi-layered defensive tactics to avoid or discourage herbivory. To the detriment of plants, herbivores, in turn, evolved intricate strategies to find, eat, and successfully digest essential plant parts to raise their own offspring. In this battle the digestive tract is the arena determining final victory or defeat as measured by growth or starvation of the herbivore. Earlier, specific molecular opponents were identified as proteases and inhibitors: digestive proteases of herbivores evolved structural motifs to occlude plant protease inhibitors, or alternatively, the insects evolved proteases capable of specifically degrading the host plant inhibitors. In response plant inhibitors evolved hyper-variable and novel protein folds to remain active against potential herbivores. At the level of protease regulation in herbivorous insects, it was shown that inhibition-insensitive digestive proteases are up-regulated when sensitive proteases are inhibited. The way this regulation operates in mammals is known as negative feedback by gut-luminal factors, so-called 'monitor peptides' that are sensitive to the concentration of active enzymes. We propose that regulation of gut enzymes by endogenous luminal factors has been an open invitation to plants to "hijack" this regulation by evolving receptor antagonists, although yet these plant factors have not been identified. In future research the question of the co-evolution of insect proteases and plant inhibitors should, therefore, be better approached from a systems level keeping in mind that evolution is fundamentally opportunistic and that the plant's fitness is primarily improved by lowering the availability of essential amino acids to an herbivore by any available mechanism.

  19. Evaluation and demonstration of remediation alternatives for historical mine waste using ash and alkaline by products; Utvaerdering och demonstration av efterbehandlingsalternativ foer historiskt gruvavfall med aska och alkaliska restprodukter

    Energy Technology Data Exchange (ETDEWEB)

    Baeckstroem, Mattias; Sartz, Lotta; Karlsson, Stefan [MTM, Man-Technology-Envionrment, Oerebro Univ., 701 82 Oerebro (Sweden)

    2009-03-15

    The results clearly show that the use of alkaline by products can significantly reduce the leakage of trace metals from historical acid mine waste. Under ideal conditions (laboratory experiments) pH increase significantly and the trace metal concentrations decrease with around 99% compared to the untreated reference. During more realistic conditions (pilot scale) the same increase in pH was not obtained and thus the decrease in trace metal concentrations was not as great. In the stabilisation experiments pH was between 5.8 and 6.8 while the trace metal reduction was around 96-99%. In the filter experiments a median pH between 4 (aged ash) and 10 (lime kiln dust) was obtained after the alkaline section. Average metal reduction is around 95% for cadmium, copper and lead while it is slightly lower for zinc (85%). In summary it is indicated that hydroxide dominated materials work best in aerated environments while carbonate dominated materials work best in reducing environments. In summary it can be concluded that the use of alkaline by products to neutralise acidic mine waste and acid mine drainage from historical mine sites give rise to both environmental and economical benefits and should therefore be encouraged as a sustainable remediation method

  20. Irrigation water acidification to neutralize alkalinity for nursery crop production: Substrate pH, electrical conductivity, nutrient concentrations, and plant nutrition and growth

    Science.gov (United States)

    Liming agents in irrigation water, typically associated with carbonates and bicarbonates of calcium and magnesium, contribute to water alkalinity. Repeated application of LA to container crops can cause media-solution pH to rise overtime, that uncorrected, can lead to a nutrient availability imbalan...

  1. Bacillus subtilis NhaC, an Na+/H+ antiporter, influences expression of the phoPR operon and production of alkaline phosphatases

    NARCIS (Netherlands)

    Pragai, Z; Eschevins, C; Bron, S; Harwood, CR

    When Bacillus subtilis is subjected to phosphate starvation, genes of the Pho regulon are either induced or repressed. Among those induced are genes encoding alkaline phosphatases (APases). A set of isogenic mutants, with a beta -galactosidase gene transcriptionally fused to the inactivated target

  2. Irrigation water acidification to neutralize alkalinity for nursery crop production: Substrate pH, electrical conductivity, and nutrient concentrations; and plant nutrition and growth

    Science.gov (United States)

    Liming agents (LA) in irrigation water, typically associated with carbonates and bicarbonates of calcium (Ca) and magnesium (Mg), contribute to water alkalinity. Repeated application of LA to container crops can cause media-solution pH to rise overtime, that uncorrected, can lead to a nutrient avail...

  3. Autoprocessing of human immunodeficiency virus type 1 protease miniprecursor fusions in mammalian cells

    Directory of Open Access Journals (Sweden)

    Chen Chaoping

    2010-07-01

    Full Text Available Abstract Background HIV protease (PR is a virus-encoded aspartic protease that is essential for viral replication and infectivity. The fully active and mature dimeric protease is released from the Gag-Pol polyprotein as a result of precursor autoprocessing. Results We here describe a simple model system to directly examine HIV protease autoprocessing in transfected mammalian cells. A fusion precursor was engineered encoding GST fused to a well-characterized miniprecursor, consisting of the mature protease along with its upstream transframe region (TFR, and small peptide epitopes to facilitate detection of the precursor substrate and autoprocessing products. In HEK 293T cells, the resulting chimeric precursor undergoes effective autoprocessing, producing mature protease that is rapidly degraded likely via autoproteolysis. The known protease inhibitors Darunavir and Indinavir suppressed both precursor autoprocessing and autoproteolysis in a dose-dependent manner. Protease mutations that inhibit Gag processing as characterized using proviruses also reduced autoprocessing efficiency when they were introduced to the fusion precursor. Interestingly, autoprocessing of the fusion precursor requires neither the full proteolytic activity nor the majority of the N-terminal TFR region. Conclusions We suggest that the fusion precursors provide a useful system to study protease autoprocessing in mammalian cells, and may be further developed for screening of new drugs targeting HIV protease autoprocessing.

  4. The Inflammatory Actions of Coagulant and Fibrinolytic Proteases in Disease

    Directory of Open Access Journals (Sweden)

    Michael Schuliga

    2015-01-01

    Full Text Available Aside from their role in hemostasis, coagulant and fibrinolytic proteases are important mediators of inflammation in diseases such as asthma, atherosclerosis, rheumatoid arthritis, and cancer. The blood circulating zymogens of these proteases enter damaged tissue as a consequence of vascular leak or rupture to become activated and contribute to extravascular coagulation or fibrinolysis. The coagulants, factor Xa (FXa, factor VIIa (FVIIa, tissue factor, and thrombin, also evoke cell-mediated actions on structural cells (e.g., fibroblasts and smooth muscle cells or inflammatory cells (e.g., macrophages via the proteolytic activation of protease-activated receptors (PARs. Plasmin, the principle enzymatic mediator of fibrinolysis, also forms toll-like receptor-4 (TLR-4 activating fibrin degradation products (FDPs and can release latent-matrix bound growth factors such as transforming growth factor-β (TGF-β. Furthermore, the proteases that convert plasminogen into plasmin (e.g., urokinase plasminogen activator evoke plasmin-independent proinflammatory actions involving coreceptor activation. Selectively targeting the receptor-mediated actions of hemostatic proteases is a strategy that may be used to treat inflammatory disease without the bleeding complications of conventional anticoagulant therapies. The mechanisms by which proteases of the coagulant and fibrinolytic systems contribute to extravascular inflammation in disease will be considered in this review.

  5. Comparison of the genomes and transcriptomes associated with the different protease secretions of Aspergillus oryzae 100-8 and 3.042.

    Science.gov (United States)

    Zhao, Guozhong; Yao, Yunping; Hou, Lihua; Wang, Chunling; Cao, Xiaohong

    2014-10-01

    Aspergillus oryzae is used to produce traditional fermented foods and beverages. A. oryzae 3.042 produces a neutral protease and an alkaline protease but rarely an acid protease, which is unfavourable to soy-sauce fermentation. A. oryzae 100-8 was obtained by N(+) ion implantation mutagenesis of A. oryzae 3.042, and the protease secretions of these two strains are different. Sequencing the genome of A. oryzae 100-8 and comparing it to the genomes of A. oryzae 100-8 and 3.042 revealed some differences, such as single nucleotide polymorphisms, nucleotide deletion or insertion. Some of these differences may reflect the ability of A. oryzae to secrete proteases. Transcriptional sequencing and analysis of the two strains during the same growth processes provided further insights into the genes and pathways involved in protease secretion.

  6. Complexing power of hydro-soluble degradation products from γ-irradiated polyvinylchloride. Influence on Eu(OH)_3(s) solubility and Eu(III) speciation in neutral to alkaline environment

    International Nuclear Information System (INIS)

    Reiller, Pascal E.; Badji, Hawa; Tabarant, Michel; Vercouter, Thomas; Fromentin, Elodie; Ferry, Muriel; Dannoux-Papin, Adeline

    2017-01-01

    The complexing power of hydrosoluble degradation products (HDPs) from an alkaline hydrolysis of a 10 MGy γ-irradiated polyvinylchloride is studied. The complexation of Eu(III), as an analogue of lanthanide and actinide radionuclides at their +III oxidation state for oxygen containing functions, is evidenced both from the increasing of Eu(OH)_3(s) dissolution, and from a complexometric titration by time-resolved luminescence spectroscopy. The dissolution of Eu(OH)_3(s) in a simplified alkaline solution (0.3 M KOH/0.1 M NaOH) increases moderately, but significantly, with the HDPs concentration. The luminescence signal of the supernatant clearly indicates the presence of several complexed Eu(III) species. Performing a complexometric titration of Eu(III) from pH 6 by alkaline HDPs shows the formation of two different species with increasing HDPs' concentration and pH. Operational complexation constants - based on dissolved carbon concentration - are proposed. The analyses of the spectra and luminescence decays seem to confirm the presence of two different species.

  7. Complexing power of hydro-soluble degradation products from γ-irradiated polyvinylchloride. Influence on Eu(OH){sub 3}(s) solubility and Eu(III) speciation in neutral to alkaline environment

    Energy Technology Data Exchange (ETDEWEB)

    Reiller, Pascal E.; Badji, Hawa; Tabarant, Michel; Vercouter, Thomas [CEA, Paris-Saclay Univ., Gif-sur-Yvette (France). Service d' Etudes Analytiques et de Reactivite des Surfaces (SEARS); Fromentin, Elodie; Ferry, Muriel [CEA, Paris-Saclay Univ., Gif-sur-Yvette (France). Service d' Etudes du Comportement des Radionucleides (SECR); Dannoux-Papin, Adeline [CEA, Bagnols-sur-Ceze (France). Service des Procedes de Decontamination et d' Enrobage

    2017-10-01

    The complexing power of hydrosoluble degradation products (HDPs) from an alkaline hydrolysis of a 10 MGy γ-irradiated polyvinylchloride is studied. The complexation of Eu(III), as an analogue of lanthanide and actinide radionuclides at their +III oxidation state for oxygen containing functions, is evidenced both from the increasing of Eu(OH){sub 3}(s) dissolution, and from a complexometric titration by time-resolved luminescence spectroscopy. The dissolution of Eu(OH){sub 3}(s) in a simplified alkaline solution (0.3 M KOH/0.1 M NaOH) increases moderately, but significantly, with the HDPs concentration. The luminescence signal of the supernatant clearly indicates the presence of several complexed Eu(III) species. Performing a complexometric titration of Eu(III) from pH 6 by alkaline HDPs shows the formation of two different species with increasing HDPs' concentration and pH. Operational complexation constants - based on dissolved carbon concentration - are proposed. The analyses of the spectra and luminescence decays seem to confirm the presence of two different species.

  8. Alkaline earth metals

    International Nuclear Information System (INIS)

    Brown, Paul L.; Ekberg, Christian

    2016-01-01

    The beryllium ion has a relatively small ionic radius. As a consequence of this small size, its hydrolysis reactions begin to occur at a relatively low pH. To determine the stability and solubility constants, however, the Gibbs energy of the beryllium ion is required. In aqueous solution calcium, like the other alkaline earth metals, only exists as a divalent cation. The size of the alkaline earth cations increases with increasing atomic number, and the calcium ion is bigger than the magnesium ion. The hydrolysis of barium(II) is weaker than that of strontium(II) and also occurs in quite alkaline pH solutions, and similarly, only the species barium hydroxide has been detected. There is only a single experimental study on the hydrolysis of radium. As with the stability constant trend, it would be expected that the enthalpy of radium would be lower than that of barium due to the larger ionic radius.

  9. Acid transformation of bauxite residue: Conversion of its alkaline characteristics

    OpenAIRE

    Kong, X.; Li, M.; Xue, S.; Hartley, W.; Chen, C.; Wu, C.; Li, X.; Li, Y.

    2016-01-01

    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...

  10. An antibiotic, heavy metal resistant and halotolerant Bacillus cereus SIU1 and its thermoalkaline protease

    Directory of Open Access Journals (Sweden)

    Vikram Surendra

    2010-07-01

    Full Text Available Abstract Background Many workers have reported halotolerant bacteria from saline conditions capable of protease production. However, antibiotic resistance and heavy metal tolerance pattern of such organisms is not documented very well. Similarly, only a few researchers have reported the pattern of pH change of fermentation medium during the course of protease production. In this study, we have isolated a halotolerant Bacillus cereus SIU1 strain from a non-saline environment and studied its antibiotic and heavy metal resistance pattern. The isolate produces a thermoalkaline protease and changes the medium pH during the course of fermentation. Thermostability of protease was also studied for 30 min. Results Seventy bacterial strains isolated from the soils of Eastern Uttar Pradesh, India were screened for protease production. All of them exhibited protease activity. However, 40% bacterial isolates were found good protease producers as observed by caseinolytic zones on milk agar plates. Among them, culture S-4 was adjudged as the best protease producer, and was identified as Bacillus cereus by morphological, biochemical and 16 S rDNA sequence analyses. The isolate was resistant to heavy metals (As2+, Pb2+, Cs1+ and antibiotics (penicillin, lincomycin, cloxacillin, pefloxacin. Its growth behavior and protease production was studied at 45°C and pH 9.0. The protease units of 88 ml-1 were noted in unoptimized modified glucose yeast extract (GYE medium during early stationary phase at 20 h incubation period. The enzyme was stable in the temperature range of 35°-55°C. Conclusions An antibiotic and heavy metal resistant, halotolerant Bacillus cereus isolate is capable of producing thermoalkaline protease, which is active and stable at pH 9.0 and 35°-55°C. This isolate may be useful in several industrial applications owing to its halotolerance and antibiotic and heavy metal resistance characteristics.

  11. Developing a new production host from a blueprint: Bacillus pumilus as an industrial enzyme producer.

    Science.gov (United States)

    Küppers, Tobias; Steffen, Victoria; Hellmuth, Hendrik; O'Connell, Timothy; Bongaerts, Johannes; Maurer, Karl-Heinz; Wiechert, Wolfgang

    2014-03-24

    Since volatile and rising cost factors such as energy, raw materials and market competitiveness have a significant impact on the economic efficiency of biotechnological bulk productions, industrial processes need to be steadily improved and optimized. Thereby the current production hosts can undergo various limitations. To overcome those limitations and in addition increase the diversity of available production hosts for future applications, we suggest a Production Strain Blueprinting (PSB) strategy to develop new production systems in a reduced time lapse in contrast to a development from scratch.To demonstrate this approach, Bacillus pumilus has been developed as an alternative expression platform for the production of alkaline enzymes in reference to the established industrial production host Bacillus licheniformis. To develop the selected B. pumilus as an alternative production host the suggested PSB strategy was applied proceeding in the following steps (dedicated product titers are scaled to the protease titer of Henkel's industrial production strain B. licheniformis at lab scale): Introduction of a protease production plasmid, adaptation of a protease production process (44%), process optimization (92%) and expression optimization (114%). To further evaluate the production capability of the developed B. pumilus platform, the target protease was substituted by an α-amylase. The expression performance was tested under the previously optimized protease process conditions and under subsequently adapted process conditions resulting in a maximum product titer of 65% in reference to B. licheniformis protease titer. In this contribution the applied PSB strategy performed very well for the development of B. pumilus as an alternative production strain. Thereby the engineered B. pumilus expression platform even exceeded the protease titer of the industrial production host B. licheniformis by 14%. This result exhibits a remarkable potential of B. pumilus to be the

  12. Effects of Protease Addition and Replacement of Soybean Meal by Corn Gluten Meal on the Growth of Broilers and on the Environmental Performances of a Broiler Production System in Greece

    Science.gov (United States)

    Bonos, Eleftherios; Anestis, Vasileios; Filioussis, Georgios; Papanastasiou, Dimitrios K.; Bartzanas, Thomas; Papaioannou, Nikolaos; Tzora, Athina; Skoufos, Ioannis

    2017-01-01

    An experimental study was conducted to examine the combined effects of adding a dietary protease, reducing the levels of soybean meal (SBM) and introducing corn gluten meal (CGM) in the ration of a group of broilers reared on a commercial Greek farm. Five hundred forty chicks were divided into three dietary treatments with six replicates of thirty birds each. The first group (Control) was fed a conventional diet based on corn and soybean meal, containing 21% w/w crude protein (CP). The second group (Soy-Prot) was supplied a corn and SBM-based diet containing a lower level of CP (20% w/w) and 200 mg of the protease RONOZYME® Proact per kg of feed. The third group (Gluten-Prot) was fed a diet without soybean-related constituents which was based on corn and CGM and with CP and protease contents identical to those of the diet of the Soy-Prot group. Body weight, feed intake, feed conversion ratio (FCR), intestinal microbiota populations and morphology, meat quality and cost were evaluated. Furthermore, a partial life cycle assessment (LCA) was performed in order to assess the potential environmental performance of the systems defined by these three dietary treatments and identify their environmental hot-spots. The growth performance of the broilers supplied the Soy-Prot diet was similar to the broilers supplied the Control diet. However, the broilers which were fed the Gluten-Prot diet at the end of the trial showed a tendency (P≤0.010) for lower weight gain and feed intake compared to those of the Control diet. When compared to the Control group, lower counts of C. perfringens (P≤0.05) were detected in the ileum and cecum parts, and lower counts of F. necrophorum (P≤0.001) were detected in the cecum part of the birds from the Gluten-Prot group. The evaluation of intestinal morphometry showed that the villus height and crypt depth values were not significantly different (P>0.05) among the experimental groups for the duodenum, jejunum and ileum parts. No

  13. Effects of Protease Addition and Replacement of Soybean Meal by Corn Gluten Meal on the Growth of Broilers and on the Environmental Performances of a Broiler Production System in Greece.

    Directory of Open Access Journals (Sweden)

    Ilias Giannenas

    Full Text Available An experimental study was conducted to examine the combined effects of adding a dietary protease, reducing the levels of soybean meal (SBM and introducing corn gluten meal (CGM in the ration of a group of broilers reared on a commercial Greek farm. Five hundred forty chicks were divided into three dietary treatments with six replicates of thirty birds each. The first group (Control was fed a conventional diet based on corn and soybean meal, containing 21% w/w crude protein (CP. The second group (Soy-Prot was supplied a corn and SBM-based diet containing a lower level of CP (20% w/w and 200 mg of the protease RONOZYME® Proact per kg of feed. The third group (Gluten-Prot was fed a diet without soybean-related constituents which was based on corn and CGM and with CP and protease contents identical to those of the diet of the Soy-Prot group. Body weight, feed intake, feed conversion ratio (FCR, intestinal microbiota populations and morphology, meat quality and cost were evaluated. Furthermore, a partial life cycle assessment (LCA was performed in order to assess the potential environmental performance of the systems defined by these three dietary treatments and identify their environmental hot-spots. The growth performance of the broilers supplied the Soy-Prot diet was similar to the broilers supplied the Control diet. However, the broilers which were fed the Gluten-Prot diet at the end of the trial showed a tendency (P≤0.010 for lower weight gain and feed intake compared to those of the Control diet. When compared to the Control group, lower counts of C. perfringens (P≤0.05 were detected in the ileum and cecum parts, and lower counts of F. necrophorum (P≤0.001 were detected in the cecum part of the birds from the Gluten-Prot group. The evaluation of intestinal morphometry showed that the villus height and crypt depth values were not significantly different (P>0.05 among the experimental groups for the duodenum, jejunum and ileum parts. No

  14. Effects of Protease Addition and Replacement of Soybean Meal by Corn Gluten Meal on the Growth of Broilers and on the Environmental Performances of a Broiler Production System in Greece.

    Science.gov (United States)

    Giannenas, Ilias; Bonos, Eleftherios; Anestis, Vasileios; Filioussis, Georgios; Papanastasiou, Dimitrios K; Bartzanas, Thomas; Papaioannou, Nikolaos; Tzora, Athina; Skoufos, Ioannis

    2017-01-01

    An experimental study was conducted to examine the combined effects of adding a dietary protease, reducing the levels of soybean meal (SBM) and introducing corn gluten meal (CGM) in the ration of a group of broilers reared on a commercial Greek farm. Five hundred forty chicks were divided into three dietary treatments with six replicates of thirty birds each. The first group (Control) was fed a conventional diet based on corn and soybean meal, containing 21% w/w crude protein (CP). The second group (Soy-Prot) was supplied a corn and SBM-based diet containing a lower level of CP (20% w/w) and 200 mg of the protease RONOZYME® Proact per kg of feed. The third group (Gluten-Prot) was fed a diet without soybean-related constituents which was based on corn and CGM and with CP and protease contents identical to those of the diet of the Soy-Prot group. Body weight, feed intake, feed conversion ratio (FCR), intestinal microbiota populations and morphology, meat quality and cost were evaluated. Furthermore, a partial life cycle assessment (LCA) was performed in order to assess the potential environmental performance of the systems defined by these three dietary treatments and identify their environmental hot-spots. The growth performance of the broilers supplied the Soy-Prot diet was similar to the broilers supplied the Control diet. However, the broilers which were fed the Gluten-Prot diet at the end of the trial showed a tendency (P≤0.010) for lower weight gain and feed intake compared to those of the Control diet. When compared to the Control group, lower counts of C. perfringens (P≤0.05) were detected in the ileum and cecum parts, and lower counts of F. necrophorum (P≤0.001) were detected in the cecum part of the birds from the Gluten-Prot group. The evaluation of intestinal morphometry showed that the villus height and crypt depth values were not significantly different (P>0.05) among the experimental groups for the duodenum, jejunum and ileum parts. No

  15. Cysteine Protease (Capparin from Capsules of Caper (Capparis spinosa

    Directory of Open Access Journals (Sweden)

    Yasar Demir

    2008-01-01

    Full Text Available Proteases are enzymes that perform very important functions in organisms and are used for a variety of objectives in vitro. In recent years, proteases have been used for clinical, pharmaceutical (alimentary digestion, anti-inflammatory, etc. and industrial applications (cheese production, meat tenderizing, leather tanning. In this research, a protease has been purified from capsules of caper (Capparis spinosa and characterized. Caper plants have been used for food and medicine since ancient times. The plant grows abundantly in certain regions of Turkey. Ammonium sulphate fractionation and a CM Sephadex column were used for purification of the enzyme. The purification enzyme has an optimum pH=5.0 and its optimum temperature was 60 °C. The vmax and Km values determined by Lineweaver-Burk graphics were 1.38 μg/(L·min and 0.88 μg/L, respectively. The purification degree and the molecular mass of the enzyme (46 kDa were determined by SDS-PAGE and gel filtration chromatography. It was investigated whether the purified and characterized protease could cause milk to congeal or digest chicken and cow meat. The results show that protease can be used for industrial production.

  16. Plant proteases for bioactive peptides release: A review.

    Science.gov (United States)

    Mazorra-Manzano, M A; Ramírez-Suarez, J C; Yada, R Y

    2017-04-10

    Proteins are a potential source of health-promoting biomolecules with medical, nutraceutical, and food applications. Nowadays, bioactive peptides production, its isolation, characterization, and strategies for its delivery to target sites are a matter of intensive research. In vitro and in vivo studies regarding the bioactivity of peptides has generated strong evidence of their health benefits. Dairy proteins are considered the richest source of bioactive peptides, however proteins from animal and vegetable origin also have been shown to be important sources. Enzymatic hydrolysis has been the process most commonly used for bioactive peptide production. Most commercial enzymatic preparations frequently used are from animal (e.g., trypsin and pepsin) and microbial (e.g., Alcalase® and Neutrase®) sources. Although the use of plant proteases is still relatively limited to papain and bromelain from papaya and pineapple, respectively, the application of new plant proteases is increasing. This review presents the latest knowledge in the use and diversity of plant proteases for bioactive peptides release from food proteins including both available commercial plant proteases as well as new potential plant sources. Furthermore, the properties of peptides released by plant proteases and health benefits associated in the control of disorders such as hypertension, diabetes, obesity, and cancer are reviewed.

  17. Investigating Mechanisms of Alkalinization for Reducing Primary Breast Tumor Invasion

    Directory of Open Access Journals (Sweden)

    Ian F. Robey

    2013-01-01

    Full Text Available The extracellular pH (pHe of many solid tumors is acidic as a result of glycolytic metabolism and poor perfusion. Acidity promotes invasion and enhances metastatic potential. Tumor acidity can be buffered by systemic administration of an alkaline agent such as sodium bicarbonate. Tumor-bearing mice maintained on sodium bicarbonate drinking water exhibit fewer metastases and survive longer than untreated controls. We predict this effect is due to inhibition of tumor invasion. Reducing tumor invasion should result in fewer circulating tumor cells (CTCs. We report that bicarbonate-treated MDA-MB-231 tumor-bearing mice exhibited significantly lower numbers of CTCs than untreated mice (. Tumor pHe buffering may reduce optimal conditions for enzymes involved in tumor invasion such as cathepsins and matrix metalloproteases (MMPs. To address this, we tested the effect of transient alkalinization on cathepsin and MMP activity using enzyme activatable fluorescence agents in mice bearing MDA-MB-231 mammary xenografts. Transient alkalinization significantly reduced the fluorescent signal of protease-specific activatable agents in vivo (. Alkalinization, however, did not affect expression of carbonic anhydrase IX (CAIX. The findings suggest a possible mechanism in a live model system for breast cancer where systemic alkalinization slows the rate of invasion.

  18. Application of protease technology in dermatology: rationale for incorporation into skin care with initial observations on formulations designed for skin cleansing, maintenance of hydration, and restoration of the epidermal permeability barrier.

    Science.gov (United States)

    Del Rosso, James Q

    2013-06-01

    This article reviews background on proteases and their functions, their physiological significance in skin, and the potential implications of incorporating specific proteases and protease blends into dermatological products, including skin care formulations. The history of protease blend formulations used in wound model studies and for other disorders is reviewed. In vitro data with use of a specific 3-protease blend with evaluation of the impact on various skin proteins and peptides is also discussed in this article.

  19. Application of Protease Technology in Dermatology: Rationale for Incorporation into Skin Care with Initial Observations on Formulations Designed for Skin Cleansing, Maintenance of Hydration, and Restoration of the Epidermal Permeability Barrier

    OpenAIRE

    Del Rosso, James Q.

    2013-01-01

    This article reviews background on proteases and their functions, their physiological significance in skin, and the potential implications of incorporating specific proteases and protease blends into dermatological products, including skin care formulations. The history of protease blend formulations used in wound model studies and for other disorders is reviewed. In vitro data with use of a specific 3-protease blend with evaluation of the impact on various skin proteins and peptides is also ...

  20. Characterization of detergent compatible protease from halophilic Virgibacillus sp. CD6.

    Science.gov (United States)

    Lam, Ming Quan; Nik Mut, Nik Nurhidayu; Thevarajoo, Suganthi; Chen, Sye Jinn; Selvaratnam, Chitra; Hussin, Huszalina; Jamaluddin, Haryati; Chong, Chun Shiong

    2018-02-01

    A halophilic bacterium, Virgibacillus sp. strain CD6, was isolated from salted fish and its extracellular protease was characterized. Protease production was found to be highest when yeast extract was used as nitrogen source for growth. The protease exhibited stability at wide range of salt concentration (0-12.5%, w/v), temperatures (20-60 °C), and pH (4-10) with maximum activity at 10.0% (w/v) NaCl, 60 °C, pH 7 and 10, indicating its polyextremophilicity. The protease activity was enhanced in the presence of Mg 2+ , Mn 2+ , Cd 2+ , and Al 3+ (107-122% relative activity), and with retention of activity > 80% for all of other metal ions examined (K + , Ca 2+ , Cu 2+ , Co 2+ , Ni 2+ , Zn 2+ , and Fe 3+ ). Both PMSF and EDTA inhibited protease activity, denoting serine protease and metalloprotease properties, respectively. High stability (> 70%) was demonstrated in the presence of organic solvents and detergent constituents, and the extracellular protease from strain CD6 was also found to be compatible in commercial detergents. Proteinaceous stain removal efficacy revealed that crude protease of strain CD6 could significantly enhance the performance of commercial detergent. The protease from Virgibacillus sp. strain CD6 could serve as a promising alternative for various applications, especially in detergent industry.

  1. Identification of an archaeal presenilin-like intramembrane protease.

    Science.gov (United States)

    Torres-Arancivia, Celia; Ross, Carolyn M; Chavez, Jose; Assur, Zahra; Dolios, Georgia; Mancia, Filippo; Ubarretxena-Belandia, Iban

    2010-09-29

    The GXGD-type diaspartyl intramembrane protease, presenilin, constitutes the catalytic core of the γ-secretase multi-protein complex responsible for activating critical signaling cascades during development and for the production of β-amyloid peptides (Aβ) implicated in Alzheimer's disease. The only other known GXGD-type diaspartyl intramembrane proteases are the eukaryotic signal peptide peptidases (SPPs). The presence of presenilin-like enzymes outside eukaryots has not been demonstrated. Here we report the existence of presenilin-like GXGD-type diaspartyl intramembrane proteases in archaea. We have employed in vitro activity assays to show that MCMJR1, a polytopic membrane protein from the archaeon Methanoculleus marisnigri JR1, is an intramembrane protease bearing the signature YD and GXGD catalytic motifs of presenilin-like enzymes. Mass spectrometry analysis showed MCMJR1 could cleave model intramembrane protease substrates at several sites within their transmembrane region. Remarkably, MCMJR1 could also cleave substrates derived from the β-amyloid precursor protein (APP) without the need of protein co-factors, as required by presenilin. Two distinct cleavage sites within the transmembrane domain of APP could be identified, one of which coincided with Aβ40, the predominant site processed by γ-secretase. Finally, an established presenilin and SPP transition-state analog inhibitor could inhibit MCMJR1. Our findings suggest that a primitive GXGD-type diaspartyl intramembrane protease from archaea can recapitulate key biochemical properties of eukaryotic presenilins and SPPs. MCMJR1 promises to be a more tractable, simpler system for in depth structural and mechanistic studies of GXGD-type diaspartyl intramembrane proteases.

  2. Identification of an archaeal presenilin-like intramembrane protease.

    Directory of Open Access Journals (Sweden)

    Celia Torres-Arancivia

    Full Text Available BACKGROUND: The GXGD-type diaspartyl intramembrane protease, presenilin, constitutes the catalytic core of the γ-secretase multi-protein complex responsible for activating critical signaling cascades during development and for the production of β-amyloid peptides (Aβ implicated in Alzheimer's disease. The only other known GXGD-type diaspartyl intramembrane proteases are the eukaryotic signal peptide peptidases (SPPs. The presence of presenilin-like enzymes outside eukaryots has not been demonstrated. Here we report the existence of presenilin-like GXGD-type diaspartyl intramembrane proteases in archaea. METHODOLOGY AND PRINCIPAL FINDINGS: We have employed in vitro activity assays to show that MCMJR1, a polytopic membrane protein from the archaeon Methanoculleus marisnigri JR1, is an intramembrane protease bearing the signature YD and GXGD catalytic motifs of presenilin-like enzymes. Mass spectrometry analysis showed MCMJR1 could cleave model intramembrane protease substrates at several sites within their transmembrane region. Remarkably, MCMJR1 could also cleave substrates derived from the β-amyloid precursor protein (APP without the need of protein co-factors, as required by presenilin. Two distinct cleavage sites within the transmembrane domain of APP could be identified, one of which coincided with Aβ40, the predominant site processed by γ-secretase. Finally, an established presenilin and SPP transition-state analog inhibitor could inhibit MCMJR1. CONCLUSIONS AND SIGNIFICANCE: Our findings suggest that a primitive GXGD-type diaspartyl intramembrane protease from archaea can recapitulate key biochemical properties of eukaryotic presenilins and SPPs. MCMJR1 promises to be a more tractable, simpler system for in depth structural and mechanistic studies of GXGD-type diaspartyl intramembrane proteases.

  3. Contemporary protease inhibitors and cardiovascular risk

    DEFF Research Database (Denmark)

    Lundgren, Jens; Mocroft, Amanda; Ryom, Lene

    2018-01-01

    PURPOSE OF REVIEW: To review the evidence linking use of HIV protease inhibitors with excess risk of cardiovascular disease (CVD) in HIV+ populations. RECENT FINDINGS: For the two contemporary most frequently used protease inhibitors, darunavir and atazanavir [both pharmacologically boosted...

  4. Human eosinophils constitutively express a unique serine protease, PRSS33.

    Science.gov (United States)

    Toyama, Sumika; Okada, Naoko; Matsuda, Akio; Morita, Hideaki; Saito, Hirohisa; Fujisawa, Takao; Nakae, Susumu; Karasuyama, Hajime; Matsumoto, Kenji

    2017-07-01

    Eosinophils play important roles in asthma, especially airway remodeling, by producing various granule proteins, chemical mediators, cytokines, chemokines and proteases. However, protease production by eosinophils is not fully understood. In the present study, we investigated the production of eosinophil-specific proteases/proteinases by transcriptome analysis. Human eosinophils and other cells were purified from peripheral blood by density gradient sedimentation and negative/positive selections using immunomagnetic beads. Protease/proteinase expression in eosinophils and release into the supernatant were evaluated by microarray analysis, qPCR, ELISA, flow cytometry and immunofluorescence staining before and after stimulation with eosinophil-activating cytokines and secretagogues. mRNAs for extracellular matrix proteins in human normal fibroblasts were measured by qPCR after exposure to recombinant protease serine 33 (PRSS33) protein (rPRSS33), created with a baculovirus system. Human eosinophils expressed relatively high levels of mRNA for metalloproteinase 25 (MMP25), a disintegrin and metalloprotease 8 (ADAM8), ADAM10, ADAM19 and PRSS33. Expression of PRSS33 was the highest and eosinophil-specific. PRSS33 mRNA expression was not affected by eosinophil-activating cytokines. Immunofluorescence staining showed that PRSS33 was co-localized with an eosinophil granule protein. PRSS33 was not detected in the culture supernatant of eosinophils even after stimulation with secretagogues, but its cell surface expression was increased. rPRSS33 stimulation of human fibroblasts increased expression of collagen and fibronectin mRNAs, at least in part via protease-activated receptor-2 activation. Activated eosinophils may induce fibroblast extracellular matrix protein synthesis via cell surface expression of PRSS33, which would at least partly explain eosinophils' role(s) in airway remodeling. Copyright © 2017 Japanese Society of Allergology. Production and hosting by Elsevier

  5. Characterization and milk coagulating properties of Cynanchum otophyllum Schneid. proteases.

    Science.gov (United States)

    Luo, Jie; Xiao, Chen; Zhang, Hao; Ren, Fazheng; Lei, Xingen; Yang, Zibiao; Yu, Zhengquan

    2018-04-01

    The herbaceous plant Cynanchum otophyllum Schneid. is widely used as a milk coagulant to make a Chinese traditional milk product, milk cake. However, the milk-clotting compounds and their mechanism remain unclear. In this study, crude proteases were extracted from the dried leaves of Cynanchum otophyllum Schneid. using citric acid-phosphate buffer and then partially purified by weak anion exchange chromatography. Two proteases, QA and QC, with molecular weights of 14 and 27 kDa, respectively, were shown to exhibit milk-clotting activity. A study of the effects of pH and temperature on the milk-clotting activity and proteolytic activity of the proteases showed that they exhibited good pH stability from pH 5.5 to 7.5 and good thermal stability at temperatures from 50 to 70°C. The QA and QC were the cysteine proteases, able to hydrolyze β-casein and κ-casein completely, and α-casein partially. The cleavage site on κ-casein determined by Orbitrap (Thermo Fisher Scientific, San Jose, CA) analysis showed that QA and QC could cleave κ-casein at Ser132-Thr133. Overall, the results suggest that the Cynanchum otophyllum Schneid. proteases are a promising milk-clotting enzyme that could be used for manufacturing milk cake and cheese. Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  6. Synthesis of glycinamides using protease immobilized magnetic nanoparticles

    Directory of Open Access Journals (Sweden)

    Abha Sahu

    2016-12-01

    Full Text Available In the present investigation, Bacillus subtilis was isolated from slaughterhouse waste and screened for the production of protease enzyme. The purified protease was successfully immobilized on magnetic nanoparticles (MNPs and used for the synthesis of series of glycinamides. The binding and thermal stability of protease on MNPs was confirmed by FTIR spectroscopy and TGA analysis. The surface morphology of MNPs before and after protease immobilization was carried out using SEM analysis. XRD pattern revealed no phase change in MNPs after enzyme immobilization. The processing parameters for glycinamides synthesis viz. temperature, pH, and time were optimized using Response Surface Methodology (RSM by using Design Expert (9.0.6.2. The maximum yield of various amides 2 butyramidoacetic acid (AMD-1,83.4%, 2-benzamidoacetic acid (AMD-2,80.5% and 2,2′((carboxymethyl amino-2-oxoethyl-2-hydroxysuccinylbis(azanediyldiacetic acid (AMD-3,80.8% formed was observed at pH-8, 50 °C and 30 min. The synthesized immobilized protease retained 70% of the initial activity even after 8 cycles of reuse.

  7. 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.

  8. Hydrogen storage alloy electrode for metal-hydride alkaline storage battery its production method; Kinzoku-suisokabutsu aruakri chikudenchiyo no suiso kyuzo gokin denkyoku oyobi sono seizo hoho

    Energy Technology Data Exchange (ETDEWEB)

    Matsuura, Y.; Nogami, K.; Kimoto, M.; Higashiyama, N.; Kuroda, Y.; Yonezu, I.; Nishio, K.

    1997-03-28

    Recently, it is proposed to employ the hydrogen storage alloy produced by means of rapidly solidifying single roll method, i.e., a method of projecting the molten alloy onto the surface of roll rotating in high speed as for the negative electrode material of the metal hydride alkaline battery. However, the hydrogen storage alloy produced by the single roll method has a heterogeneous grain size. So that the utilization of the hydrogen storage alloy is limited. This invention solves the problem. The rare earth-nickel system hydrogen storage alloy ribbon with average thickness of 0.08 - 0.35 mm is produced by means of single roll method. The grain size of the alloy is over 0.2 micrometer on roll surface side and below 20 micrometers on open surface side. The above said alloy is ground to average particle size of 25 - 70 micrometers to be used for the hydrogen absorbent. In this way, the metal hydride alkaline battery with excellent high rate discharge characteristic at the initial stage of charge-discharge cycle, excellent charge-discharge cycle characteristic, and excellent inner pressure characteristic can be produced. 2 figs., 5 tabs.

  9. Hydrogen storage alloy electrode of metal hydride alkaline storage battery and its production method; Kinzoku-suisokabutsu arukari chikudenchiyo no suiso kyuzo gokin denkyoku oyobi sono seizo hoho

    Energy Technology Data Exchange (ETDEWEB)

    Matsuura, Y.; Nogami, K.; Kimoto, M.; Higashiyama, N.; Kuroda, Y.; Yonezu, I.; Nishio, K.

    1997-03-28

    Recently, a proposal was made of employing the hydrogen storage alloy produced by means f rapidly solidifying single roll method, i.e., a method of projecting the molten alloy onto the surface of roll rotating in high speed as for the negative electrode material of the metal hydride alkaline battery. However, the hydrogen storage alloy produced by the single roll method has a heterogeneous grain size. This invention solves the problem. The Mm{center_dot}Ni{center_dot}Co{center_dot}Al{center_dot}Mn alloy ribbon with average thickness of 0.08 - 0.35 mm is produced by means of single roll method. The grain size of the alloy is over 0.2 micrometer on roll surface side and below 18 micrometers on open surface side. The alloy is ground to be used for the hydrogen absorbent. The general formula of this alloy is MmR(x) (Mm = mischmetal, R = Ni, Co, Al, Mn). In this way, the metal hydride alkaline battery with excellent high rate discharge characteristic at the initial stage of charge-discharge cycle, excellent charge-discharge cycle characteristic, and excellent inner pressure characteristic can be produced. 2 figs., 5 tabs.

  10. Isolation of protease producing novel Bacillus cereus and detection ...

    African Journals Online (AJOL)

    user

    2011-02-14

    Feb 14, 2011 ... Key words: Protease, production, optimization, Bacillus sp. INTRODUCTION ... Nutrient broth (5 g peptone and 3 g meat extract, pH 7.0, Merck) was used as the common growth ... nitrate through nitrite. It was determined that ...

  11. Milk Clotting Activity of Protease, Extracted from Rhizome of Taffin ...

    African Journals Online (AJOL)

    MBI

    2017-03-07

    Mar 7, 2017 ... The increasing prices of calf rennets, their accessibility and ethical concerns ... the region with a massive annual production (FAO, ... valuable group of enzymes with various industrial ... use of protease enzymes in the food industry .... In the procedure, Bovine Serum Albumin ..... Agricultural Economics.

  12. Physical and chemical properties of the acid protease from ...

    African Journals Online (AJOL)

    samsung

    2016-03-02

    Mar 2, 2016 ... is the principal set of biochemical changes during ... coagulating ability by analysis of the products of casein ... for protease activity, milk-clotting activity and protein content. ..... Figure 5, the content of casein components decreased in .... Purification, caracterization, molecular cloning and modelling of its.

  13. Factor VII-activating protease

    DEFF Research Database (Denmark)

    Ramanathan, Ramshanker; Gram, Jørgen B; Sand, Niels Peter R

    2017-01-01

    : Factor VII-activating protease (FSAP) may regulate development of cardiovascular disease (CVD). We evaluated sex differences in FSAP measures and examined the association between FSAP and coronary artery calcification (CAC) in a middle-aged population. Participants were randomly selected citizens...

  14. Carbohydrase and protease supplementation increased ...

    African Journals Online (AJOL)

    A trial was conducted to evaluate whether the addition of commercial enzyme preparations containing carbohydrases and a protease would increase the available metabolizable energy (ME) of maize-soya-based broiler diets. Seven thousand five hundred and sixty (7560) day-old Ross 788 chicks were randomly allocated ...

  15. Alkaline solution/binder ratio as a determining factor in the alkaline activation of aluminosilicates

    Energy Technology Data Exchange (ETDEWEB)

    Ruiz-Santaquiteria, C., E-mail: ruiz.cs@ietcc.csic.es [Eduardo Torroja Institute (CSIC), c/Serrano Galvache, n Degree-Sign 4, 28033 Madrid (Spain); Skibsted, J. [Instrument Centre for Solid-State NMR Spectroscopy, Interdisciplinary Nanoscience Center (iNANO), Department of Chemistry, Aarhus University, DK-8000 Aarhus C (Denmark); Fernandez-Jimenez, A.; Palomo, A. [Eduardo Torroja Institute (CSIC), c/Serrano Galvache, n Degree-Sign 4, 28033 Madrid (Spain)

    2012-09-15

    This study investigates the effect of the alkaline solution/binder (S/B) ratio on the composition and nanostructure of the reaction products generated in the alkaline activation of aluminosilicates. The experiments used two mixtures of fly ash and dehydroxylated white clay and for each of these, varying proportions of the solution components. The alkali activator was an 8 M NaOH solution (with and without sodium silicate) used at three S/B ratios: 0.50, 0.75 and 1.25. The {sup 29}Si, {sup 27}Al MAS NMR and XRD characterisation of the reaction products reveal that for ratios nearest the value delivering suitable paste workability, the reaction-product composition and structure depend primarily on the nature and composition of the starting materials and the alkaline activator used. However, when an excess alkaline activator is present in the system, the reaction products tend to exhibit SiO{sub 2}/Al{sub 2}O{sub 3} ratios of approximately 1, irrespective of the composition of the starting binder or the alkaline activator.

  16. Increased river alkalinization in the Eastern U.S.

    Science.gov (United States)

    Kaushal, Sujay S; Likens, Gene E; Utz, Ryan M; Pace, Michael L; Grese, Melissa; Yepsen, Metthea

    2013-09-17

    The interaction between human activities and watershed geology is accelerating long-term changes in the carbon cycle of rivers. We evaluated changes in bicarbonate alkalinity, a product of chemical weathering, and tested for long-term trends at 97 sites in the eastern United States draining over 260,000 km(2). We observed statistically significant increasing trends in alkalinity at 62 of the 97 sites, while remaining sites exhibited no significant decreasing trends. Over 50% of study sites also had statistically significant increasing trends in concentrations of calcium (another product of chemical weathering) where data were available. River alkalinization rates were significantly related to watershed carbonate lithology, acid deposition, and topography. These three variables explained ~40% of variation in river alkalinization rates. The strongest predictor of river alkalinization rates was carbonate lithology. The most rapid rates of river alkalinization occurred at sites with highest inputs of acid deposition and highest elevation. The rise of alkalinity in many rivers throughout the Eastern U.S. suggests human-accelerated chemical weathering, in addition to previously documented impacts of mining and land use. Increased river alkalinization has major environmental implications including impacts on water hardness and salinization of drinking water, alterations of air-water exchange of CO2, coastal ocean acidification, and the influence of bicarbonate availability on primary production.

  17. Characterization of the Protease Activity of Detergents: Laboratory Practicals for Studying the Protease Profile and Activity of Various Commercial Detergents

    Science.gov (United States)

    Valls, Cristina; Pujadas, Gerard; Garcia-Vallve, Santi; Mulero, Miquel

    2011-01-01

    Detergent enzymes account for about 30% of the total worldwide production of enzymes and are one of the largest and most successful applications of modern industrial biotechnology. Proteases can improve the wash performance of household, industrial, and institutional laundry detergents used to remove protein-based stains such as blood, grass, body…

  18. An efficient method to eliminate the protease activity contaminating commercial bovine pancreatic DNase I.

    Science.gov (United States)

    Le, Tien; Lee, Hak Jin; Jin, Hyung Jong

    2015-08-15

    A method was developed to eliminate the proteases contaminating commercial DNase I, which can cause degradation of target protein during the purification process. Bio Basic DNase stock solution (in Tris-HCl buffer [pH 8.0] containing 5mM CaCl2) was first incubated at 50 °C to generate autolysis of proteases and zymogens, leading to a significant reduction in protease activity while preserving DNase activity. The residual protease activity was completely inhibited by further incubation with 2mM PMSF (phenylmethylsulfonyl fluoride) or 2× S8830 inhibitor cocktail. This approach could be readily applicable to eliminate the protease activity in any DNase products or during the preparation of commercial DNase. Copyright © 2015 Elsevier Inc. All rights reserved.

  19. An enzyme from the earthworm Eisenia fetida is not only a protease but also a deoxyribonuclease.

    Science.gov (United States)

    Pan, Rong; Zhou, Yuan; He, Hai-Jin; He, Rong-Qiao

    2011-04-01

    The earthworm enzyme Eisenia fetida Protease-III-1 (EfP-III-1) is known as a trypsin-like protease which is localized in the alimentary canal of the earthworm. Here, we show that EfP-III-1 also acts as a novel deoxyribonuclease. Unlike most DNases, this earthworm enzyme recognizes 5'-phosphate dsDNA (5'P DNA) and degrades it without sequence specificity, but does not recognize 5'OH DNA. As is the case for most DNases, Mg(2+) was observed to markedly enhance the DNase activity of EfP-III-1. Whether the earthworm enzyme functioned as a DNase or as a protease depended on the pH values of the enzyme solution. The protein acted as a protease under alkaline conditions whereas it exhibited DNase activity under acid conditions. At pH 7.0, the enzyme could work as either a DNase or a protease. Given the complex living environment of the earthworm, this dual function of EfP-III-1 may play an important role in the alimentary digestion of the earthworm. Copyright © 2011 Elsevier Inc. All rights reserved.

  20. Differential Response of Extracellular Proteases of Trichoderma Harzianum Against Fungal Phytopathogens.

    Science.gov (United States)

    Sharma, Vivek; Salwan, Richa; Sharma, Prem N

    2016-09-01

    In the present study, production of extracellular proteases by Trichoderma harzianum was evaluated based on the relative gene expression and spectrophotometric assay. The fungal isolates were grown in Czapek Dox Broth medium supplemented with deactivated mycelium of plant fungal pathogens such as Fusarium oxysporum, Colletotrichum capsici, Gloeocercospora sorghi, and Colletotrichum truncatum. The maximum protease activity was detected after 48 h of incubation against Colletotrichum spp. Similarly in qRT-PCR, the relative gene expression of four proteases varied from 48 to 96 h against host pathogens in a time-independent manner. Among proteases, statistically significant upregulation of asp, asp, and srp was observed against Colletotrichum spp., followed by F. oxysporum. But in the case of pepM22, maximum upregulation was observed against F. oxysporum. The variation in enzyme assay and qRT-PCR of proteases at different time intervals against various fungal phytopathogens could be due to the limitation of using casein as a substrate for all types of proteases or protease-encoding transcripts selected for qRT-PCR, which may not be true representative of total protease activity.

  1. Purification and characterization of an extracellular haloalkaline serine protease from the moderately halophilic bacterium, Bacillus iranensis (X5B).

    Science.gov (United States)

    Ghafoori, Hossein; Askari, Mansoure; Sarikhan, Sajjad

    2016-03-01

    This study reports the purification and characterization of an extracellular haloalkaline serine protease from the moderately halophilic bacterium, Bacillus iranensis, strain X5B. The enzyme was purified to homogeneity by acetone precipitation, ultrafiltration and carboxymethyl (CM) cation exchange chromatography, respectively. The purified protease was a monomeric enzyme with a relative molecular mass of 48-50 kDa and it was inhibited by PMSF indicating that it is a serine-protease. The optimum pH, temperature and NaCl concentration were 9.5, 35 °C and 0.98 M, respectively. The enzyme showed a significant tolerance to salt and alkaline pH. It retained approximately 50% of activity at 2.5 M NaCl and about 70% of activity at highly alkaline pH of 11.0; therefore, it was a moderately halophilic and also can be activated by metals, especially by Ca(2+). The specific activity of the purified protease was measured to be 425.23 μmol of tyrosine/min per mg of protein using casein as a substrate. The apparent K m and V max values were 0.126 mM and 0.523 mM/min, respectively and the accurate value of k cat was obtained as 3.284 × 10(-2) s(-1). These special and important characteristics make this serine protease as valuable tool for industrial applications.

  2. 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.

  3. 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.

  4. Influence of initial temperature and heating method in the temperature profile during alkaline dissolution of Al for the production of Mo-99

    Energy Technology Data Exchange (ETDEWEB)

    Camilo, Ruth L.; Araujo, Izilda C.; Mindrisz, Ana C.; Forbicini, Christina A.L.G. de O., E-mail: rcamilo@ipen.br, E-mail: cruzaraujo22@gmail.com, E-mail: acmindri@ipen.br, E-mail: cforbici@ipen.br [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil)

    2013-07-01

    Radionuclides in nuclear medicine can be used for diagnosis and therapy. The {sup 99m}Tc, son of {sup 99}Mo, is most often used in nuclear medicine as tracer element because of its favorable nuclear properties, accounting for about 80% of all diagnostic procedures in vivo. Aiming to resolve the dependency of Brazil with respect to the supply of {sup 99}Mo was created the Brazilian Multipurpose Reactor project (BMR), started in 2008, having as main objective to produce about 1000 Ci/week of {sup 99}Mo. This study is part of the project to obtain {sup 9}'9Mo by alkaline dissolution of UAl{sub x}-Al targets. The initial reaction temperature is an important parameter, since it has great influence on the value of the maximum temperature and dissolution time. According to literature, for security reasons the dissolution process must have its temperature controlled so that the maximum temperature has to be around 90 deg C. The behavior of the temperature during dissolution using three different methods of heating in order to minimize the fluctuation of temperature during dissolution, keeping its maximum value at around 90 deg C was studied. The three methods of heating chosen were: a) initial temperature of 85 deg C with continuous heating, b) heating water bath until it reaches the initial temperature (70 to 95 deg C), turning off after that, and c) external heating until it reached the starting temperature (60-95 deg C). The alkaline solution used was 3 mol.L{sup -1} NaOH{sub 3} and 2 mol.L{sup -1} NaNO{sub 3}. In the first study it was observed that after 1 minute of dissolution the solution temperature reached 100 deg C on average, up to a maximum of 109 deg C, ending with values around 95 deg C. In the second study after 3 minutes of dissolution the maximum temperature was 106 deg C and the minimum 100 deg C. In the third study the temperature rise during dissolution increased with increasing initial temperature which practically remains constant until the end

  5. Purification and biochemical characterization of a 22-kDa stable cysteine- like protease from the excretory-secretory product of the liver fluke Fasciola hepatica by using conventional techniques.

    Science.gov (United States)

    Hemici, Ahmed; Benerbaiha, Roumaila Sabrina; Bendjeddou, Dalila

    2017-11-15

    This study describes the purification and characterization of a stable protease activity isolated from Fasciola hepatica adult worms maintained in vitro by employing acetone precipitation (40-60%) followed by a gel filtration through Sephadex G-100 and DEAE- cellulose ion exchange column. Through this three-step purification, the enzyme was purified 11-fold with a specific activity of 1893.9U/mg and 31.5% recovery. After the final ultrafiltration step, the purification fold was increased up to 13.1 and the overall activity yield reached a rate of 18.8%. The MW of the purified protease was estimated by reducing SDS-PAGE to be 22kDa while the proteolytic activity detection was carried out by zymography on non-denaturing SDS-PAGE containing the casein as substrate. Using this substrate, the protease showed extreme proteolytic activity at pH 5.5 and temperature 35-40°C and was highly stable over a wide range of pH, from 5.0 to 10.0. In addition to its preference for the Z-Phe-Arg-AMC fluorogenic substrate resulting in maximum proteolytic activity (99.7%) at pH 7.0, the pure protease exhibited highest cleavage activity against hemoglobin and casein substrates at pH 5.5 (85.6% and 82.8%, respectively). The K m values obtained for this protease were 5.4, 13, 160 and approximately 1000μM using respectively the fluorogenic substrate Z-Phe-Arg-AMC, hemoglobin, casein and albumin. The protease activity was completely inhibited either by E-64 inhibitor (5mM) or iodoacetamide (10mM), indicating its cysteine nature. The usefulness of the purified protease as an antigen was studied by immunoblotting. Thus, sera from sheep experimentally infected with F. hepatica recognized the protease band at 2 weeks post-infection (WPI) and strongly at 7 WPI. The early detection of antibodies anti- F. hepatica suggests the application of this molecule as a specific epitope for the serodiagnosis of fascioliasis disease. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. Cysteine Protease Zymography: Brief Review.

    Science.gov (United States)

    Wilkesman, Jeff

    2017-01-01

    Cysteine proteases play multiple roles in basically all aspects of physiology and development. In plants, they are involved in growth and development and in accumulation and mobilization of storage proteins. Furthermore, they are engaged in signalling pathways and in the response to biotic and abiotic stresses. In animals and also in humans, they are responsible for senescence and apoptosis, prohormone processing, and ECM remodelling. When analyzed by zymography, the enzyme must be renaturated after SDS-PAGE. SDS must be washed out and substituted by Triton X-100. Gels are then further incubated under ideal conditions for activity detection. Cysteine proteases require an acidic pH (5.0-6.0) and a reducing agent, usually DTT. When screening biological samples, there is generally no previous clue on what peptidase class will be present, neither optimal proteolysis conditions are known. Hence, it is necessary to assess several parameters, such as incubation time, pH, temperature, influence of ions or reducing agents, and finally evaluate the inhibition profile. For detection of cysteine peptidase activity, the use of specific inhibitors, such as E-64, can be used to prevent the development of cysteine peptidase activity bands and positively confirm its presence. Here four different protocols to assess cysteine protease activity from different sources are presented.

  7. Alkalinity of the Mediterranean Sea

    OpenAIRE

    Schneider, Anke; Wallace, Douglas W.R.; Körtzinger, Arne

    2007-01-01

    Total alkalinity (AT) was measured during the Meteor 51/2 cruise, crossing the Mediterranean Sea from west to east. AT concentrations were high (∼2600 μmol kg−1) and alkalinity-salinity-correlations had negative intercepts. These results are explained by evaporation coupled with high freshwater AT inputs into coastal areas. Salinity adjustment of AT revealed excess alkalinity throughout the water column compared to mid-basin surface waters. Since Mediterranean waters are supersaturated with r...

  8. 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.

  9. Molecular characterization of protease activity in Serratia sp. strain SCBI and its importance in cytotoxicity and virulence.

    Science.gov (United States)

    Petersen, Lauren M; Tisa, Louis S

    2014-11-01

    A newly recognized Serratia species, termed South African Caenorhabditis briggsae isolate (SCBI), is both a mutualist of the nematode Caenorhabditis briggsae KT0001 and a pathogen of lepidopteran insects. Serratia sp. strain SCBI displays high proteolytic activity, and because secreted proteases are known virulence factors for many pathogens, the purpose of this study was to identify genes essential for extracellular protease activity in Serratia sp. strain SCBI and to determine what role proteases play in insect pathogenesis and cytotoxicity. A bank of 2,100 transposon mutants was generated, and six SCBI mutants with defective proteolytic activity were identified. These mutants were also defective in cytotoxicity. The mutants were found defective in genes encoding the following proteins: alkaline metalloprotease secretion protein AprE, a BglB family transcriptional antiterminator, an inosine/xanthosine triphosphatase, GidA, a methyl-accepting chemotaxis protein, and a PIN domain protein. Gene expression analysis on these six mutants showed significant downregulation in mRNA levels of several different types of predicted protease genes. In addition, transcriptome sequencing (RNA-seq) analysis provided insight into how inactivation of AprE, GidA, and a PIN domain protein influences motility and virulence, as well as protease activity. Using quantitative reverse transcription-PCR (qRT-PCR) to further characterize expression of predicted protease genes in wild-type Serratia sp. SCBI, the highest mRNA levels for the alkaline metalloprotease genes (termed prtA1 to prtA4) occurred following the death of an insect host, while two serine protease and two metalloprotease genes had their highest mRNA levels during active infection. Overall, these results indicate that proteolytic activity is essential for cytotoxicity in Serratia sp. SCBI and that its regulation appears to be highly complex. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  10. Degradation of cellulosic materials under the alkaline conditions of a cementitious repository for low- and intermediate level radioactive waste. Pt. III. Effect of degradation products on the sorption of radionuclides on feldspar

    International Nuclear Information System (INIS)

    Loon, L.R. van; Glaus, M.A.; Laube, A.; Stallone, S.

    1999-01-01

    The effect of degradation products of different cellulosic materials on the sorption behaviour of Th(IV), Eu(III) and Ni(II) on feldspar at pH 13.3 was studied. For all three metals, a decrease in sorption could be observed with increasing concentration of organics in solution. For Th(IV), α-ISA is the effective ligand present in the solutions of degraded cellulose, independent on the type of cellulose studied. For Eu(III), α-ISA is the effective ligand in the case of pure cellulose degradation. In the case of other cellulosic materials, unknown ligands cause the sorption reduction. For Ni(II), also unknown ligands cause sorption reduction, independent on the type of cellulose studied. These unknown ligands are not formed during alkaline degradation of cellulose, but are present as impurities in certain cellulosic materials. (orig.)

  11. Identification of SlpB, a Cytotoxic Protease from Serratia marcescens.

    Science.gov (United States)

    Shanks, Robert M Q; Stella, Nicholas A; Hunt, Kristin M; Brothers, Kimberly M; Zhang, Liang; Thibodeau, Patrick H

    2015-07-01

    The Gram-negative bacterium and opportunistic pathogen Serratia marcescens causes ocular infections in healthy individuals. Secreted protease activity was characterized from 44 ocular clinical isolates, and a higher frequency of protease-positive strains was observed among keratitis isolates than among conjunctivitis isolates. A positive correlation between protease activity and cytotoxicity to human corneal epithelial cells in vitro was determined. Deletion of prtS in clinical keratitis isolate K904 reduced, but did not eliminate, cytotoxicity and secreted protease production. This indicated that PrtS is necessary for full cytotoxicity to ocular cells and implied the existence of another secreted protease(s) and cytotoxic factors. Bioinformatic analysis of the S. marcescens Db11 genome revealed three additional open reading frames predicted to code for serralysin-like proteases noted here as slpB, slpC, and slpD. Induced expression of prtS and slpB, but not slpC and slpD, in strain PIC3611 rendered the strain cytotoxic to a lung carcinoma cell line; however, only prtS induction was sufficient for cytotoxicity to a corneal cell line. Strain K904 with deletion of both prtS and slpB genes was defective in secreted protease activity and cytotoxicity to human cell lines. PAGE analysis suggests that SlpB is produced at lower levels than PrtS. Purified SlpB demonstrated calcium-dependent and AprI-inhibited protease activity and cytotoxicity to airway and ocular cell lines in vitro. Lastly, genetic analysis indicated that the type I secretion system gene, lipD, is required for SlpB secretion. These genetic data introduce SlpB as a new cytotoxic protease from S. marcescens. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  12. Uncoupling of Protease trans-Cleavage and Helicase Activities in Pestivirus NS3.

    Science.gov (United States)

    Zheng, Fengwei; Lu, Guoliang; Li, Ling; Gong, Peng; Pan, Zishu

    2017-11-01

    The nonstructural protein NS3 from the Flaviviridae family is a multifunctional protein that contains an N-terminal protease and a C-terminal helicase, playing essential roles in viral polyprotein processing and genome replication. Here we report a full-length crystal structure of the classical swine fever virus (CSFV) NS3 in complex with its NS4A protease cofactor segment (PCS) at a 2.35-Å resolution. The structure reveals a previously unidentified ∼2,200-Å 2 intramolecular protease-helicase interface comprising three clusters of interactions, representing a "closed" global conformation related to the NS3-NS4A cis -cleavage event. Although this conformation is incompatible with protease trans -cleavage, it appears to be functionally important and beneficial to the helicase activity, as the mutations designed to perturb this conformation impaired both the helicase activities in vitro and virus production in vivo Our work reveals important features of protease-helicase coordination in pestivirus NS3 and provides a key basis for how different conformational states may explicitly contribute to certain functions of this natural protease-helicase fusion protein. IMPORTANCE Many RNA viruses encode helicases to aid their RNA genome replication and transcription by unwinding structured RNA. Being naturally fused to a protease participating in viral polyprotein processing, the NS3 helicases encoded by the Flaviviridae family viruses are unique. Therefore, how these two enzyme modules coordinate in a single polypeptide is of particular interest. Here we report a previously unidentified conformation of pestivirus NS3 in complex with its NS4A protease cofactor segment (PCS). This conformational state is related to the protease cis -cleavage event and is optimal for the function of helicase. This work provides an important basis to understand how different enzymatic activities of NS3 may be achieved by the coordination between the protease and helicase through different

  13. Alkaline Activator Impact on the Geopolymer Binders

    Science.gov (United States)

    Błaszczyński, Tomasz Z.; Król, Maciej R.

    2017-10-01

    Concrete structures are constantly moving in the direction of improving the durability. Durability depends on many factors, which are the composition of concrete mix, the usage of additives and admixtures and the place, where material will work and carry the load. The introduction of new geopolymer binders for geopolymer structures adds a new aspect that is type of used activator. This substance with strongly alkaline reaction is divided because of the physical state, the alkaline degree and above all the chemical composition. Taking into account, that at present the geopolymer binders are made essentially from waste materials or by-products from the combustion of coal or iron ore smelting, unambiguous determination of the effect of the activator on the properties of the geopolymer material requires a number of trials, researches and observation. This paper shows the influence of the most alkaline activators on the basic parameters of the durability of geopolymer binders. In this study there were used highly alkaline hydroxides, water glasses and granules, which are waste materials in a variety of processes taking place in chemical plants. As the substrate of geopolymer binders there were used fly ash which came from coal and high calcareous ash from the burning of lignite.

  14. TiO2-NT electrodes modified with Ag and diamond like carbon (DLC) for hydrogen production by alkaline water electrolysis

    Science.gov (United States)

    Baran, Evrim; Baz, Zeynep; Esen, Ramazan; Yazici Devrim, Birgül

    2017-10-01

    In present work, the two-step anodization technique was applied for synthesis of TiO2 nanotube (NT). Silver and diamond like carbon (DLC) were coated on the surface of as prepared TiO2-NT using chemical reduction method and MW ECR plasma system. The morphology, composition and structure of the electrodes were examined by field emission scanning electron microscopy (FE-SEM), energy dispersive X-ray spectroscopy (EDX) and X-ray diffraction (XRD). The results showed that Ag nanoparticles, having size in the range of 48-115 nm, are evenly distributed on the top, inside and outside surface of TiO2-NT and when DLC was coated on the surface of TiO2-NT and TiO2-NT-Ag, the top of nanotubes were partially open and the pore diameter of hexagonal structure decreased from 165 nm to of 38-80 nm. On the other hand, the microhardness test and contact angle measurements revealed that additions of Ag and diamond like carbon have a positive effect on the mechanical properties of TiO2-NT film. The electrocatalytic properties of the electrodes towards the hydrogen evolution reaction (HER) were investigated by the electrochemical measurements recorded in 1 M KOH solution. In addition, long-term durability of electrodes towards HER and the energy consumption of alkaline electrolysis were investigated. The energy requirement showed that while the deposition of silver provides approximately 14.95% savings of the energy consumption, the DLC coating causes increase in energy consumption.

  15. Temperature Dependence of Mineral Solubility in Water. Part 2. Alkaline and Alkaline Earth Bromides

    Science.gov (United States)

    Krumgalz, B. S.

    2018-03-01

    Databases of alkaline and alkaline earth bromide solubilities in water at various temperatures were created using experimental data from publications over about the last two centuries. Statistical critical evaluation of the created databases was produced since there were enough independent data sources to justify such evaluation. The reliable experimental data were adequately described by polynomial expressions over various temperature ranges. Using the Pitzer approach for ionic activity and osmotic coefficients, the thermodynamic solubility products for the discussed bromide minerals have been calculated at various temperature intervals and also represented by polynomial expressions.

  16. 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.

  17. Reduction of proteinuria through podocyte alkalinization.

    Science.gov (United States)

    Altintas, Mehmet M; Moriwaki, Kumiko; Wei, Changli; Möller, Clemens C; Flesche, Jan; Li, Jing; Yaddanapudi, Suma; Faridi, Mohd Hafeez; Gödel, Markus; Huber, Tobias B; Preston, Richard A; Jiang, Jean X; Kerjaschki, Dontscho; Sever, Sanja; Reiser, Jochen

    2014-06-20

    Podocytes are highly differentiated cells and critical elements for the filtration barrier of the kidney. Loss of their foot process (FP) architecture (FP effacement) results in urinary protein loss. Here we show a novel role for the neutral amino acid glutamine in structural and functional regulation of the kidney filtration barrier. Metabolic flux analysis of cultured podocytes using genetic, toxic, and immunologic injury models identified increased glutamine utilization pathways. We show that glutamine uptake is increased in diseased podocytes to couple nutrient support to increased demand during the disease state of FP effacement. This feature can be utilized to transport increased amounts of glutamine into damaged podocytes. The availability of glutamine determines the regulation of podocyte intracellular pH (pHi). Podocyte alkalinization reduces cytosolic cathepsin L protease activity and protects the podocyte cytoskeleton. Podocyte glutamine supplementation reduces proteinuria in LPS-treated mice, whereas acidification increases glomerular injury. In summary, our data provide a metabolic opportunity to combat urinary protein loss through modulation of podocyte amino acid utilization and pHi. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. 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).

  19. Method of cleaning alkaline metal

    International Nuclear Information System (INIS)

    Kawakami, Yukio; Naito, Kesahiro; Iizawa, Katsuyuki; Nakasuji, Takashi

    1981-01-01

    Purpose: To prevent scattering of used sodium and aqueous alkaline solution when cleaning used sodium and metallic sodium adhering to equipment with an aqueous alkaline solution. Method: A sodium treating container is filled with an aqueous alkaline solution, and stainless steel gauze is sunk in the container. Equipment to be cleaned such as equipment with sodium adhering to it are retained under the gauze and are thus cleaned. On the other hand, the surface of the aqueous alkaline solution is covered with a fluid paraffin liquid covering material. Thus, the hydrogen produced by the reaction of the sodium and the aqueous alkaline solution will float up, pass through the liquid covering material and be discharged. The sodium will pass through the gauze and float upwardly while reacting with the aqueous alkaline solution in a partic ulate state to the boundary between the aqueous alkaline solution and up to the covering material, and thus the theratment reaction will continue. Thus, the cover material prevents the sodium and the aqueous alkaline solution from scattering. (Kamimura, M.)

  20. Perspectives de développement de la production industrielle d'hydrogène par électrolyse alcaline avancée Development Outlook for Industrial Hydrogen Production by Advanced Alkaline Electrolysis

    Directory of Open Access Journals (Sweden)

    Derive C.

    2006-11-01

    -prototypes sont prolongés et deux programmes complémentaires d'essais sur les composants principaux sont actuellement menés avant l'engagement de la phase de qualification de l'électrolyseur industriel sur un pilote de 2 MWe. Under the development conditions of the French nuclear program, which has succeeded in producing electricity at an interesting cost in off-peak hours, hydrogen production by water electrolysis can be considered in the mediumterm to be in competition with other hydrogen production processes such as natural-gas reforming. Since 1976 Electricité de France (EDF and Gaz de France (GDF have been cooperating on an R & D project on hydrogen production by alkaline water electrolysis with the aim of reducing the investment cost and maintaining the efficiency level compared to present-day installations. Prior research has shown that these objectives can be attained by advanced electrolysis with increased current density and temperature. These technical constraints have led EDF and GDF to undertake research on the chemical and mechanical resistance of materials, on the selection of suitable cell components, and on improving the overall design of installations. The two French industrial groups, headed by Alsthom-Atlantique and Creusot-Loire, have been associated to this research since 1979 and have set the following operating conditions:(a potash-base electrolyte (40% mass;(b temperatures of 120 and 160°C;(c pressures of 30 and 70 bar. In an initial phase, these groups made a technico-economic survey of the massive production of hydrogen by plants having a power of about 300 MWe. Detailed plans were drawn up for a 2-MWe pilot plant, and technological choices were made on 25-30 kWe prototype loops. To give further certainty to the choices made and to go further into problems of scaling up to large-size electrolyzers, tests on prototype loops were extended, and additional tests are now being made of the principal components before undertaking the qualification phase

  1. Production of enzymes by a newly isolated Bacillus sp. TMF-1 in solid state fermentation on agricultural by-products: The evaluation of substrate pretreatment methods.

    Science.gov (United States)

    Salim, Abdalla Ali; Grbavčić, Sanja; Šekuljica, Nataša; Stefanović, Andrea; Jakovetić Tanasković, Sonja; Luković, Nevena; Knežević-Jugović, Zorica

    2017-03-01

    Study on potential of different agro-industrial waste residues for supporting the growth of newly isolated Bacillus sp. TMF-1 strain under solid-state fermentation (SSF) was conducted aiming to produce several industrially valuable enzymes. Since the feasibility of the initial study was confirmed, further objectives included evaluation of several pretreatments of the studied agricultural by-products (soybean meal, sunflower meal, maize bran, maize pericarp, olive oil cake and wheat bran) on the microbial productivity as means of enhancing the yields of produced proteases, α-amylases, cellulases and pectinases. Among acid/alkaline treatment, ultrasound and microwave assisted methods, chemical treatments superiorly affected most of the studied substrates. Highest yields of produced proteases (50.5IUg -1 ) and α-amylases (50.75IUg -1 ) were achieved on alkaline treated corn pericarp. Alkaline treatment also promoted the secretion of cellulases on maize bran (1.19IUg -1 ). Highest yield of pectinases was obtained on untreated soybean meal (64.90IUg -1 ). Copyright © 2016 Elsevier Ltd. All rights reserved.

  2. A Deg-protease family protein in marine Synechococcus is involved in outer membrane protein organization

    Directory of Open Access Journals (Sweden)

    Rhona Kayra Stuart

    2014-06-01

    Full Text Available Deg-family proteases are a periplasm-associated group of proteins that are known to be involved in envelope stress responses and are found in most microorganisms. Orthologous genes SYNW2176 (in strain WH8102 and sync_2523 (strain CC9311 are predicted members of the Deg-protease family and are among the few genes induced by copper stress in both open ocean and coastal marine Synechococcus strains. In contrast to the lack of a phenotype in a similar knockout in Synechocystis PCC6803, a SYNW2176 knockout mutant in strain WH8102 was much more resistant to copper than the wild-type. The mutant also exhibited a significantly altered outer membrane protein composition which may contribute to copper resistance, longer lag phase after transfer, low-level consistent alkaline phosphatase activity, and an inability to induce high alkaline phosphatase activity in response to phosphate stress. This phenotype suggests a protein-quality-control role for SYNW2176, the absence of which leads to a constitutively activated stress response. Deg-protease family proteins in this ecologically important cyanobacterial group thus help to determine outer membrane responses to both nutrients and toxins.

  3. A recombinant fusion protein-based, fluorescent protease assay for high throughput-compatible substrate screening.

    Science.gov (United States)

    Bozóki, Beáta; Gazda, Lívia; Tóth, Ferenc; Miczi, Márió; Mótyán, János András; Tőzsér, József

    2018-01-01

    In connection with the intensive investigation of proteases, several methods have been developed for analysis of the substrate specificity. Due to the great number of proteases and the expected target molecules to be analyzed, time- and cost-efficient high-throughput screening (HTS) methods are preferred. Here we describe the development and application of a separation-based HTS-compatible fluorescent protease assay, which is based on the use of recombinant fusion proteins as substrates of proteases. The protein substrates used in this assay consists of N-terminal (hexahistidine and maltose binding protein) fusion tags, cleavage sequences of the tobacco etch virus (TEV) and HIV-1 proteases, and a C-terminal fluorescent protein (mApple or mTurquoise2). The assay is based on the fluorimetric detection of the fluorescent proteins, which are released from the magnetic bead-attached substrates by the proteolytic cleavage. The protease assay has been applied for activity measurements of TEV and HIV-1 proteases to test the suitability of the system for enzyme kinetic measurements, inhibition studies, and determination of pH optimum. We also found that denatured fluorescent proteins can be renatured after SDS-PAGE of denaturing conditions, but showed differences in their renaturation abilities. After in-gel renaturation both substrates and cleavage products can be identified by in-gel UV detection. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. Purification and characterization of protease enzyme from ...

    African Journals Online (AJOL)

    user

    2013-03-20

    Mar 20, 2013 ... Full Length Research Paper. Purification and ... ting into small peptides and free amino acids, which can ... Isolated strain was cultured in synthetic medium- casein (SMC; ... Protease activity was assayed by sigma's non-specific protease ... following buffers: 0.05 M citrate-phosphate buffer (pH 5 to 6), Tris-.

  5. Current and Novel Inhibitors of HIV Protease

    Czech Academy of Sciences Publication Activity Database

    Pokorná, Jana; Machala, L.; Řezáčová, Pavlína; Konvalinka, Jan

    2009-01-01

    Roč. 1, č. 3 (2009), s. 1209-1239 ISSN 1999-4915 R&D Projects: GA MŠk 1M0508 Grant - others:GA AV ČR(CZ) IAAX00320901 Program:IA Institutional research plan: CEZ:AV0Z40550506 Keywords : HIV protease * protease inhibitor * HAART Subject RIV: CE - Biochemistry

  6. Effect of alkaline addition on anaerobic sludge digestion with combined pretreatment of alkaline and high pressure homogenization.

    Science.gov (United States)

    Fang, Wei; Zhang, Panyue; Zhang, Guangming; Jin, Shuguang; Li, Dongyi; Zhang, Meixia; Xu, Xiangzhe

    2014-09-01

    To improve anaerobic digestion efficiency, combination pretreatment of alkaline and high pressure homogenization was applied to pretreat sewage sludge. Effect of alkaline dosage on anaerobic sludge digestion was investigated in detail. SCOD of sludge supernatant significantly increased with the alkaline dosage increase after the combined pretreatment because of sludge disintegration. Organics were significantly degraded after the anaerobic digestion, and the maximal SCOD, TCOD and VS removal was 73.5%, 61.3% and 43.5%, respectively. Cumulative biogas production, methane content in biogas and biogas production rate obviously increased with the alkaline dosage increase. Considering both the biogas production and alkaline dosage, the optimal alkaline dosage was selected as 0.04 mol/L. Relationships between biogas production and sludge disintegration showed that the accumulative biogas was mainly enhanced by the sludge disintegration. The methane yield linearly increased with the DDCOD increase as Methane yield (ml/gVS)=4.66 DDCOD-9.69. Copyright © 2014 Elsevier Ltd. All rights reserved.

  7. Improvement of shelf life of soymilk using immobilized protease of Oerskovia xanthineolytica NCIM 2839.

    Science.gov (United States)

    Sahoo, A K; Gaikwad, V S; Ranveer, R C; Dandge, P B; Waghmare, S R

    2016-12-01

    Protease enzyme has lot of commercial applications, so the cost-effective production of protease using sunflower oil seed waste was carried out from Oerskovia xanthineolyitca NCIM 2839. The maximum protease production was after 24 h of incubation with 2.5 % oil seed waste concentration. O. xanthineolytica was found to produce two proteases-P1 and P2. The proteases were purified using 60 % cold acetone precipitation and DEAE-cellulose ion exchange chromatography. SDS-PAGE revealed molecular weight of P1 and P2 was 36 and 24 kDa, respectively. P1 and P2 were optimally active at pH 7.0 and pH 7.5 at temperature 35 and 40 °C, respectively. Analysis of hydrolyzed product of P1 and P2 by HPLC reveals that the P1 has endoprotease and P2 has exoprotease activity. The treated soy milk with immobilized proteases showed increased shelf life and removal of off flavor.

  8. Thermophilic Alkaline Fermentation Followed by Mesophilic Anaerobic Digestion for Efficient Hydrogen and Methane Production from Waste-Activated Sludge: Dynamics of Bacterial Pathogens as Revealed by the Combination of Metagenomic and Quantitative PCR Analyses.

    Science.gov (United States)

    Wan, Jingjing; Jing, Yuhang; Rao, Yue; Zhang, Shicheng; Luo, Gang

    2018-03-15

    Thermophilic alkaline fermentation followed by mesophilic anaerobic digestion (TM) for hydrogen and methane production from waste-activated sludge (WAS) was investigated. The TM process was also compared to a process with mesophilic alkaline fermentation followed by a mesophilic anaerobic digestion (MM) and one-stage mesophilic anaerobic digestion (M) process. The results showed that both hydrogen yield (74.5 ml H 2 /g volatile solids [VS]) and methane yield (150.7 ml CH 4 /g VS) in the TM process were higher than those (6.7 ml H 2 /g VS and 127.8 ml CH 4 /g VS, respectively) in the MM process. The lowest methane yield (101.2 ml CH 4 /g VS) was obtained with the M process. Taxonomic results obtained from metagenomic analysis showed that different microbial community compositions were established in the hydrogen reactors of the TM and MM processes, which also significantly changed the microbial community compositions in the following methane reactors compared to that with the M process. The dynamics of bacterial pathogens were also evaluated. For the TM process, the reduced diversity and total abundance of bacterial pathogens in WAS were observed in the hydrogen reactor and were further reduced in the methane reactor, as revealed by metagenomic analysis. The results also showed not all bacterial pathogens were reduced in the reactors. For example, Collinsella aerofaciens was enriched in the hydrogen reactor, which was also confirmed by quantitative PCR (qPCR) analysis. The study further showed that qPCR was more sensitive for detecting bacterial pathogens than metagenomic analysis. Although there were some differences in the relative abundances of bacterial pathogens calculated by metagenomic and qPCR approaches, both approaches demonstrated that the TM process was more efficient for the removal of bacterial pathogens than the MM and M processes. IMPORTANCE This study developed an efficient process for bioenergy (H 2 and CH 4 ) production from WAS and elucidates the

  9. Molecular characterization of 45 kDa aspartic protease of Trichinella spiralis.

    Science.gov (United States)

    Park, Jong Nam; Park, Sang Kyun; Cho, Min Kyoung; Park, Mi-Kyung; Kang, Shin Ae; Kim, Dong-Hee; Yu, Hak Sun

    2012-12-21

    In a previous study, we identified an aspartic protease gene (Ts-Asp) from the Trichinella spiralis muscle stage larva cDNA library. The gene sequence of Ts-Asp was 1281 bp long and was found to encode a protein consisting of 405 amino acids, with a molecular mass of 45.248 kD and a pI of 5.95. The deduced Ts-Asp has a conserved catalytic motif with catalytic aspartic acid residues in the active site, a common characteristic of aspartic proteases. In addition, the deduced amino acid sequence of Ts-Asp was found to possess significant homology (above 50%) with aspartic proteases from nematode parasites. Results of phylogenetic analysis indicated a close relationship of Ts-Asp with cathepsin D aspartic proteases. For production of recombinant Ts-Asp (rTs-Asp), the pGEX4T expression system was used. Like other proteases, the purified rTs-Asp was able to digest collagen matrix in vitro. Abundant expression of Ts-Asp was observed in muscle stage larva. Ts-Asp was detected in ES proteins, and was able to elicit the production of specific antibodies. It is the first report of molecular characterization of aspartic protease isolated from T. spiralis. Copyright © 2012 Elsevier B.V. All rights reserved.

  10. Streptomyces flavogriseus HS1: isolation and characterization of extracellular proteases and their compatibility with laundry detergents.

    Science.gov (United States)

    Ghorbel, Sofiane; Kammoun, Maher; Soltana, Hala; Nasri, Moncef; Hmidet, Noomen

    2014-01-01

    The present study describes the isolation of a new protease producing Streptomyces strain HS1 and the biochemical characterization of the secreted proteases. By sequencing of its noted 16S rDNA, HS1 strain was found to have a 100% identity with Streptomyces flavogriseus. The highest protease production was found using FermII media. In these conditions maximum protease production (99 U/mL) was obtained after 96 h incubation at 30°C and 150 rpm. HS1 strain produced at least five proteases as revealed by zymogram technique. The enzyme preparation exhibited activity over a broad range of pH (5-11) and temperature (25-70°C). Optimum activity was observed at a pH of 7.0 and a temperature of 50°C. Proteolytic activity was significantly unaffected by Ca(2+) and Mg(2+). EDTA and PMSF highly decreased the original activity. The crude extracellular proteases showed high stability when used as a detergent additive. These properties offer an interesting potential for enzymatic hydrolysis at the industrial level.

  11. Advances in zymography techniques and patents regarding protease analysis.

    Science.gov (United States)

    Wilkesman, Jeff; Kurz, Liliana

    2012-08-01

    Detection of enzymatic activity on gel electrophoresis, namely zymography, is a technique that has received increasing attention in the last 10 years, according to the number of articles published. A growing amount of enzymes, mainly proteases, are now routinely detected by zymography. Detailed analytical studies are beginning to be published, as well as new patents have been developed. This new article updates the information covered in our last review, condensing the recent publications dealing with the identification of proteolytic enzymes in electrophoretic gel supports and its variations. The new advances of this method are basically focused towards two dimensional zymography and transfer zymography. Though comparatively fewer patents have been published, they basically coincide in the study of matrix metalloproteases. The tendency is foreseen to be very productive in the area of zymoproteomics, combining electrophoresis and mass spectrometry for the analysis of proteases.

  12. Natural inhibitors of tumor-associated proteases

    International Nuclear Information System (INIS)

    Magdolen, U.; Krol, J.; Sato, S.; Schmitt, M.; Magdolen, V.; Krueger, A.; Mueller, M.M.; Sperl, S.

    2002-01-01

    The turnover and remodelling of extracellular matrix (ECM) is an essential part of many normal biological processes including development, morphogenesis, and wound healing. ECM turnover also occurs in severe pathological situations like artherosclerosis, fibrosis, tumor invasion and metastasis. The major proteases involved in this turnover are serine proteases (especially the urokinase-type plasminogen activator/plasmin system), matrix metalloproteases (a family of about 20 zinc-dependent endopeptidases including collagenases, gelatinases, stromelysins, and membrane-type metalloproteases), and cysteine proteases. In vivo, the activity of these proteases is tightly regulated in the extracellular space by zymogen activation and/or controlled inhibition. In the present review, we give an overview on the structure and biochemical properties of important tumor-associated protease inhibitors such as plasminogen activator inhibitor type 1 and type 2 (PAI-1, PAI-2), tissue inhibitors of metalloproteinases (TIMP-1, -2, -3, and -4), and the cysteine protease inhibitor cystatin C. Interestingly, some of these inhibitors of tumor-associated proteases display multiple functions which rather promote than inhibit tumor progression, when the presence of inhibitors in the tumor tissue is not balanced. (author)

  13. Extracellular proteases of Trichoderma species. A review.

    Science.gov (United States)

    Kredics, L; Antal, Zsuzsanna; Szekeres, A; Hatvani, L; Manczinger, L; Vágvölgyi, Cs; Nagy, Erzsébet

    2005-01-01

    Cellulolytic, xylanolytic, chitinolytic and beta-1,3-glucanolytic enzyme systems of species belonging to the filamentous fungal genus Trichoderma have been investigated in details and are well characterised. The ability of Trichoderma strains to produce extracellular proteases has also been known for a long time, however, the proteolytic enzyme system is relatively unknown in this genus. Fortunately, in the recent years more and more attention is focused on the research in this field. The role of Trichoderma proteases in the biological control of plant pathogenic fungi and nematodes has been demonstrated, and it is also suspected that they may be important for the competitive saprophytic ability of green mould isolates and may represent potential virulence factors of Trichoderma strains as emerging fungal pathogens of clinical importance. The aim of this review is to summarize the information available about the extracellular proteases of Trichoderma. Numerous studies are available about the extracellular proteolytic enzyme profiles of Trichoderma strains and about the effect of abiotic environmental factors on protease activities. A number of protease enzymes have been purified to homogeneity and some protease encoding genes have been cloned and characterized. These results will be reviewed and the role of Trichoderma proteases in biological control as well as their advantages and disadvantages in biotechnology will be discussed.

  14. Gut proteases target Yersinia invasin in vivo

    Directory of Open Access Journals (Sweden)

    Freund Sandra

    2011-04-01

    Full Text Available Abstract Background Yersinia enterocolitica is a common cause of food borne gastrointestinal disease. After oral uptake, yersiniae invade Peyer's patches of the distal ileum. This is accomplished by the binding of the Yersinia invasin to β1 integrins on the apical surface of M cells which overlie follicle associated lymphoid tissue. The gut represents a barrier that severely limits yersiniae from reaching deeper tissues such as Peyer's patches. We wondered if gut protease attack on invasion factors could contribute to the low number of yersiniae invading Peyer's patches. Findings Here we show that invasin is rapidly degraded in vivo by gut proteases in the mouse infection model. In vivo proteolytic degradation is due to proteolysis by several gut proteases such as trypsin, α-chymotrypsin, pancreatic elastase, and pepsin. Protease treated yersiniae are shown to be less invasive in a cell culture model. YadA, another surface adhesin is cleaved by similar concentrations of gut proteases but Myf was not cleaved, showing that not all surface proteins are equally susceptible to degradation by gut proteases. Conclusions We demonstrate that gut proteases target important Yersinia virulence factors such as invasin and YadA in vivo. Since invasin is completely degraded within 2-3 h after reaching the small intestine of mice, it is no longer available to mediate invasion of Peyer's patches.

  15. 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.

  16. Antibody proteases: induction of catalytic response.

    Science.gov (United States)

    Gabibov, A G; Friboulet, A; Thomas, D; Demin, A V; Ponomarenko, N A; Vorobiev, I I; Pillet, D; Paon, M; Alexandrova, E S; Telegin, G B; Reshetnyak, A V; Grigorieva, O V; Gnuchev, N V; Malishkin, K A; Genkin, D D

    2002-10-01

    Most of the data accumulated throughout the years on investigation of catalytic antibodies indicate that their production increases on the background of autoimmune abnormalities. The different approaches to induction of catalytic response toward recombinant gp120 HIV-1 surface protein in mice with various autoimmune pathologies are described. The peptidylphosphonate conjugate containing structural part of gp120 molecule is used for reactive immunization of NZB/NZW F1, MRL, and SJL mice. The specific modification of heavy and light chains of mouse autoantibodies with Val-Ala-Glu-Glu-Glu-Val-PO(OPh)2 reactive peptide was demonstrated. Increased proteolytic activity of polyclonal antibodies in SJL mice encouraged us to investigate the production of antigen-specific catalytic antibodies on the background of induced experimental autoimmune encephalomyelitis (EAE). The immunization of autoimmune-prone mice with the engineered fusions containing the fragments of gp120 and encephalitogenic epitope of myelin basic protein (MBP(89-104)) was made. The proteolytic activity of polyclonal antibodies isolated from the sera of autoimmune mice immunized by the described antigen was shown. Specific immune response of SJL mice to these antigens was characterized. Polyclonal antibodies purified from sera of the immunized animals revealed proteolytic activity. The antiidiotypic approach to raise the specific proteolytic antibody as an "internal image" of protease is described. The "second order" monoclonal antibodies toward subtilisin Carlsberg revealed pronounced proteolytic activity.

  17. A Simple and Fast Method for the Production and Characterization of Methylic and Ethylic Biodiesels from Tucum Oil via an Alkaline Route

    Directory of Open Access Journals (Sweden)

    Marcelo Firmino de Oliveira

    2011-01-01

    Full Text Available A simple, fast, and complete route for the production of methylic and ethylic biodiesel from tucum oil is described. Aliquots of the oil obtained directly from pressed tucum (pulp and almonds were treated with potassium methoxide or ethoxide at 40°C for 40 min. The biodiesel form was removed from the reactor and washed with 0.1 M HCl aqueous solution. A simple distillation at 100°C was carried out in order to remove water and alcohol species from the biodiesel. The oxidative stability index was obtained for the tucum oil as well as the methylic and ethylic biodiesel at 6.13, 2.90, and 2.80 h, for storage times higher than 8 days. Quality control of the original oil and of the methylic and ethylic biodiesels, such as the amount of glycerin produced during the transesterification process, was accomplished by the TLC, GC-MS, and FT-IR techniques. The results obtained in this study indicate a potential biofuel production by simple treatment of tucum, an important Amazonian fruit.

  18. Protease and protease inhibitory activity in pregnant and postpartum involuting uterus

    International Nuclear Information System (INIS)

    Milwidsky, A.; Beller, U.; Palti, Z.; Mayer, M.

    1982-01-01

    The presence of two distinct proteolytic activities in the rat uterus was confirmed with 14 C-labeled globin used as a sensitive protein substrate and following release of label into the trichloroacetic acid-soluble supernatant fraction. Protease I is a cytoplasmic acid protease while protease II is associated with the pellet fraction, can be extracted by 0.6 M sodium chloride, and is active at pH 7.0. Protease I activity is low during pregnancy and markedly increases at term achieving maximal activity at day 3 post partum with a subsequent decline to preterm activity values. Lactation did not affect the uterine protease I activity. Protease II activity is not significantly different during pregnancy, at term, and post partum. The presence of an inhibitor of protease I was suggested by a decrease in enzyme activity with an increased cytosolic protein concentration. The inhibitor also lessened bovine trypsin activity but had no effect on protease II. Although its inhibitory potency on trypsin fluctuated during the various uterine physiologic stages, these changes appeared to be statistically insignificant. Human uterine samples were also found to contain the two protease activities with similar changes in protease I post partum. It is suggested that, both in the rat and in man, uterine involution post partum is associated with a marked increase in activity of acid cytosolic protease, while a particulate neutral protease and a soluble inhibitor of trypsin, which are also present in uterine cells, do not appear to play a significant role in the dissolution of uterine tissues after parturition

  19. 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.

  20. Activation of ADAM 12 protease by copper

    DEFF Research Database (Denmark)

    Loechel, F; Wewer, Ulla M.

    2001-01-01

    Conversion of latent proteases to the active form occurs by various mechanisms characteristic for different protease families. Here we report that the disintegrin metalloprotease ADAM 12-S is activated by Cu(II). Copper activation is distinct from the cysteine switch component of latency: elimina......Conversion of latent proteases to the active form occurs by various mechanisms characteristic for different protease families. Here we report that the disintegrin metalloprotease ADAM 12-S is activated by Cu(II). Copper activation is distinct from the cysteine switch component of latency......: elimination of the ADAM 12 cysteine switch by a point mutation in the propeptide had no effect on copper activation, whereas mutation of an unpaired cysteine residue in the catalytic domain resulted in a mutant form of ADAM 12-S that was insensitive to copper. This suggests a multi-step activation mechanism...... for ADAM 12 involving both furin cleavage and copper binding....

  1. Partial Purification and Characterization of Extracellular Protease ...

    African Journals Online (AJOL)

    USER

    Keywords: Protease, lactic acid bacteria, Pediococcus acidilactici, enzyme ... confers organoleptic improvements in fermented foods ... was characterized by studying the effect of substrate ... addition of solid ammonium sulphate up to 80%.

  2. Interspecific differences between D. pulex and D. magna in tolerance to cyanobacteria with protease inhibitors.

    Directory of Open Access Journals (Sweden)

    Christian J Kuster

    Full Text Available It is known that cyanobacteria negatively affect herbivores due to their production of toxins such as protease inhibitors. In the present study we investigated potential interspecific differences between two major herbivores, Daphnia magna and Daphnia pulex, in terms of their tolerance to cyanobacteria with protease inhibitors. Seven clones each of D. magna and of D. pulex were isolated from different habitats in Europe and North America. To test for interspecific differences in the daphnids' tolerance to cyanobacteria, their somatic and population growth rates were determined for each D. magna and D. pulex clone after exposure to varying concentrations of two Microcystis aeruginosa strains. The M. aeruginosa strains NIVA and PCC(- contained either chymotrypsin or trypsin inhibitors, but no microcystins. Mean somatic and population growth rates on a diet with 20% NIVA were significantly more reduced in D. pulex than in D. magna. On a diet with 10% PCC(-, the population growth of D. pulex was significantly more reduced than that of D. magna. This indicates that D. magna is more tolerant to cyanobacteria with protease inhibitors than D. pulex. The reduction of growth rates was possibly caused by an interference of cyanobacterial inhibitors with proteases in the gut of Daphnia, as many other conceivable factors, which might have been able to explain the reduced growth, could be excluded as causal factors. Protease assays revealed that the sensitivities of chymotrypsins and trypsins to cyanobacterial protease inhibitors did not differ between D. magna and D. pulex. However, D. magna exhibited a 2.3-fold higher specific chymotrypsin activity than D. pulex, which explains the observed higher tolerance to cyanobacterial protease inhibitors of D. magna. The present study suggests that D. magna may control the development of cyanobacterial blooms more efficiently than D. pulex due to differences in their tolerance to cyanobacteria with protease

  3. Detection of extracellular proteases from microorganisms on agar plates

    Directory of Open Access Journals (Sweden)

    Alane Beatriz Vermelho

    1996-12-01

    Full Text Available We present herein an improved assay for detecting the presence of extracellular proteases from microorganisms on agar plates. Using different substrates (gelatin, BSA, hemoglobin incorporated into the agar and varying the culture medium composition, we were able to detect proteolytic activities from Pseudomonas aeruginosa, Micrococcus luteus and Serratia marcescens as well as the influence that these components displayed in the expression of these enzymes. For all microorganisms tested we found that in agar-BHI or yeast extract medium containing gelatin the sensitivity of proteinase detection was considerably greater than in BSA-agar or hemoglobin-agar. However, when BSA or hemoglobin were added to the culture medium, there was an increase in growth along with a marked reduction in the amount of proteinase production. In the case of M. luteus the incorporation of glycerol in BHI or yeast extract gelatin-agar induced protease liberation. Our results indicate that the technique described here is of value for detecting extracellular proteases directly in the culture medium, by means of a qualitative assay, simple, inexpensive, straight forward method to assess the presence of the proteolytic activity of a given microorganism colony with great freedom in substrate selection.

  4. Purification and characterization of protease enzyme from ...

    African Journals Online (AJOL)

    The enzyme was active in pH range 5 to11 and temperature of 30 to 80°C. The optimum pH and the temperature for protease activity were recorded to be pH 8 and 50°C, respectively. The enzyme was stable up to 40°C and pH 9. The protease activity was inhibited by Zn2+, Ni2+ and Sn2+ and increased by Ca2+, Mg2+ ...

  5. Alkaline carbonates in blast furnace process

    Directory of Open Access Journals (Sweden)

    P. Besta

    2014-10-01

    Full Text Available The production of iron in blast furnaces is a complex of physical, chemical and mechanical processes. The input raw materials contain not only metallic components, but also a number of negative elements. The most important negative elements include alkaline carbonates. They can significantly affect the course of the blast furnace process and thus the overall performance of the furnace. As a result of that, it is essential to accurately monitor the alkali content in the blast furnace raw materials. The article analyzes the alkali content in input and output raw materials and their impact on the blast furnace process.

  6. Modelling of potentially promising SARS protease inhibitors

    International Nuclear Information System (INIS)

    Plewczynski, Dariusz; Hoffmann, Marcin; Grotthuss, Marcin von; Knizewski, Lukasz; Rychewski, Leszek; Eitner, Krystian; Ginalski, Krzysztof

    2007-01-01

    In many cases, at the beginning of a high throughput screening experiment some information about active molecules is already available. Active compounds (such as substrate analogues, natural products and inhibitors of related proteins) are often identified in low throughput validation studies on a biochemical target. Sometimes the additional structural information is also available from crystallographic studies on protein and ligand complexes. In addition, the structural or sequence similarity of various protein targets yields a novel possibility for drug discovery. Co-crystallized compounds from homologous proteins can be used to design leads for a new target without co-crystallized ligands. In this paper we evaluate how far such an approach can be used in a real drug campaign, with severe acute respiratory syndrome (SARS) coronavirus providing an example. Our method is able to construct small molecules as plausible inhibitors solely on the basis of the set of ligands from crystallized complexes of a protein target, and other proteins from its structurally homologous family. The accuracy and sensitivity of the method are estimated here by the subsequent use of an electronic high throughput screening flexible docking algorithm. The best performing ligands are then used for a very restrictive similarity search for potential inhibitors of the SARS protease within the million compounds from the Ligand.Info small molecule meta-database. The selected molecules can be passed on for further experimental validation

  7. Modelling of potentially promising SARS protease inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Plewczynski, Dariusz [Interdisciplinary Centre for Mathematical and Computational Modelling, ICM, Warsaw University, Pawinskiego 5a Street, 02-106 Warsaw (Poland); Hoffmann, Marcin [BioInfoBank Institute, Limanowskiego 24A/16, 60-744 Poznan (Poland); Grotthuss, Marcin von [BioInfoBank Institute, Limanowskiego 24A/16, 60-744 Poznan (Poland); Knizewski, Lukasz [Interdisciplinary Centre for Mathematical and Computational Modelling, ICM, Warsaw University, Pawinskiego 5a Street, 02-106 Warsaw (Poland); Rychewski, Leszek [BioInfoBank Institute, Limanowskiego 24A/16, 60-744 Poznan (Poland); Eitner, Krystian [BioInfoBank Institute, Limanowskiego 24A/16, 60-744 Poznan (Poland); Ginalski, Krzysztof [Interdisciplinary Centre for Mathematical and Computational Modelling, ICM, Warsaw University, Pawinskiego 5a Street, 02-106 Warsaw (Poland)

    2007-07-18

    In many cases, at the beginning of a high throughput screening experiment some information about active molecules is already available. Active compounds (such as substrate analogues, natural products and inhibitors of related proteins) are often identified in low throughput validation studies on a biochemical target. Sometimes the additional structural information is also available from crystallographic studies on protein and ligand complexes. In addition, the structural or sequence similarity of various protein targets yields a novel possibility for drug discovery. Co-crystallized compounds from homologous proteins can be used to design leads for a new target without co-crystallized ligands. In this paper we evaluate how far such an approach can be used in a real drug campaign, with severe acute respiratory syndrome (SARS) coronavirus providing an example. Our method is able to construct small molecules as plausible inhibitors solely on the basis of the set of ligands from crystallized complexes of a protein target, and other proteins from its structurally homologous family. The accuracy and sensitivity of the method are estimated here by the subsequent use of an electronic high throughput screening flexible docking algorithm. The best performing ligands are then used for a very restrictive similarity search for potential inhibitors of the SARS protease within the million compounds from the Ligand.Info small molecule meta-database. The selected molecules can be passed on for further experimental validation.

  8. Sample preparation in alkaline media

    International Nuclear Information System (INIS)

    Nobrega, Joaquim A.; Santos, Mirian C.; Sousa, Rafael A. de; Cadore, Solange; Barnes, Ramon M.; Tatro, Mark

    2006-01-01

    The use of tetramethylammonium hydroxide, tertiary amines and strongly alkaline reagents for sample treatment involving extraction and digestion procedures is discussed in this review. The preparation of slurries is also discussed. Based on literature data, alkaline media offer a good alternative for sample preparation involving an appreciable group of analytes in different types of samples. These reagents are also successfully employed in tailored speciation procedures wherein there is a critical dependence on maintenance of chemical forms. The effects of these reagents on measurements performed using spectroanalytical techniques are discussed. Several undesirable effects on transport and atomization processes necessitate use of the method of standard additions to obtain accurate results. It is also evident that alkaline media can improve the performance of techniques such as inductively coupled plasma mass spectrometry and accessories, such as autosamplers coupled to graphite furnace atomic absorption spectrometers

  9. Recovery of silver from used X-ray film using alkaline protease from ...

    African Journals Online (AJOL)

    Silver is an important industrial metal used in several areas such as photographic and x-ray films, jewelries, silver wares and electronic objects. Silver is used for photographic film/x-ray film because of its matchless quality as a light-sensitive material for making a photographic image. Silver is not destroyed in the ...

  10. Carbohydrate protease conjugates: Stabilized proteases for peptide synthesis

    Energy Technology Data Exchange (ETDEWEB)

    Wartchow, C.A.; Wang, Peng; Bednarski, M.D.; Callstrom, M.R. [Ohio State Univ., Columbus, OH (United States)]|[Lawrence Berkeley Lab., CA (United States)

    1995-12-31

    The synthesis of oligopeptides using stable carbohydrate protease conjugates (CPCs) was examined in acetonitrile solvent systems. CPC[{alpha}-chymotrypsin] was used for the preparation of peptides containing histidine, phenylalanine, tryptophan in the P{sub 1} position in 60-93% yield. The CPC[{alpha}-chymotrypsin]-catalyzed synthesis of octamer Z-Gly-Gly-Phe-Gly-Gly-Phe-Gly-Gly-OEt from Z-Gly-Gly-Phe-Gly-Gly-Phe-OMe was achieved in 71% yield demonstrating that synthesis peptides containing both hydrophylic and hydrophobic amino acids. The P{sub 2} specificity of papain for aromatic residues was utilized for the 2 + 3 coupling of Z-Tyr-Gly-OMe to H{sub 2}N-Gly-Phe-Leu-OH to generate the leucine enkephalin derivative in 79% yield. Although papain is nonspecific for the hydrolysis of N-benzyloxycarbonyl amino acid methyl esters in aqueous solution, the rates of synthesis for these derivitives with nucleophile leucine tert-butyl ester differed by nearly 2 orders of magnitude. CPC[thermolysin] was used to prepare the aspartame precursor Z-Asp-Phe-OMe in 90% yield. The increased stability of CPCs prepared from periodate-modified poly(2-methacryl- amido-2-deoxy-D-glucose), poly(2-methacrylamido-2-deoxy-D-galactose), and poly(5-methacryl-amido-5-deoxy-D-ribose), carbohydrate materials designed to increase the aldehyde concentration in aqueous solution, suggests that the stability of CPCs is directly related to the aldehyde concentration of the carbohydrate material. Periodate oxidation of poly(2-methacrylamido-2-deoxy-D-glucose) followed by covalent attachment to {alpha}-chymotrypsin gave a CPC with catalytic activity in potassium phosphate buffer at 90{degrees}C for 2 h. 1 fig., 1 tab., 40 refs.

  11. Comparison of four feed proteases for improvement of nutritive value of poultry feather meal

    DEFF Research Database (Denmark)

    Pedersen, Mads Brøgger; Yu, S; Plumstead, P

    2012-01-01

    in the production of cost-effective feather by-products for use as feed and fertilizers. The current study examined 4 commercial feed proteases from Bacillus subtilis, Bacillus licheniformis PWD-1, Aspergillus niger, and Serratia proteamaculans HY-3 used to hydrolyze chicken feather under different conditions...

  12. Conversion of Squid Pens to Chitosanases and Proteases via Paenibacillus sp. TKU042

    Directory of Open Access Journals (Sweden)

    Chien Thang Doan

    2018-03-01

    Full Text Available Chitosanases and proteases have received much attention due to their wide range of applications. Four kinds of chitinous materials, squid pens, shrimp heads, demineralized shrimp shells and demineralized crab shells, were used as the sole carbon and nitrogen (C/N source to produce chitosanases, proteases and α-glucosidase inhibitors (αGI by four different strains of Paenibacillus. Chitosanase productivity was highest in the culture supernatants using squid pens as the sole C/N source. The maximum chitosanase activity of fermented squid pens (0.759 U/mL was compared to that of fermented shrimp heads (0.397 U/mL, demineralized shrimp shells (0.201 U/mL and demineralized crab shells (0.216 U/mL. A squid pen concentration of 0.5% was suitable for chitosanase, protease and αGI production via Paenibacillus sp. TKU042. Multi-purification, including ethanol precipitation and column chromatography of Macro-Prep High S as well as Macro-Prep DEAE (diethylaminoethyl, led to the isolation of Paenibacillus sp. TKU042 chitosanase and protease with molecular weights of 70 and 35 kDa, respectively. For comparison, 16 chitinolytic bacteria, including strains of Paenibacillus, were investigated for the production of chitinase, exochitinase, chitosanase, protease and αGI using two kinds of chitinous sources.

  13. Studies on activity, distribution, and zymogram of protease, α-amylase, and lipase in the paddlefish Polyodon spathula.

    Science.gov (United States)

    Ji, H; Sun, H T; Xiong, D M

    2012-06-01

    A series of biochemical determination and electrophoretic observations have been conducted to analyze the activities and characteristics of protease, α-amylase, and lipase of paddlefish Polyodon spathula. The results obtained have been compared with those of bighead carp (Aristichthys nobilis) and hybrid sturgeon (Huso dauricus ♀ × Acipenser schrenki Brandt ♂), in order to increase available knowledge of the physiological characteristics of this sturgeon species and to gain information with regard to its nutrition. Further, a comparative study of enzymatic activity, distribution, and characterization between commercial feed-reared paddlefish (CG) and natural live food-reared (NG) paddlefish was conducted. Results showed that higher proteolytic activity was observed in the pH range 2.5-3.0 and at a pH of 7.0 for paddlefish. Levels of acid protease activity of paddlefish were similar to that of hybrid sturgeon, and significantly higher than that of bighead carp. The inhibition assay of paddlefish showed that the rate of inhibition of tosyl-phenylalanine chloromethyl ketone was approximately 2.6-fold that of tosyl-lysine chloromethyl ketone. There was no significant difference observed for acid protease activity between PG and CG groups, whereas the activity of alkaline protease, α-amylase, and lipase in the PG group were significantly lower than those in the CG group. The substrate sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis further showed that there were certain types of enzymes, especially α-amylase, with similar molecular mass in the paddlefish and hybrid sturgeon. It can be inferred that acid digestion was main mechanism for protein hydrolysis in paddlefish, as reported for other fishes with a stomach. This indicates that the paddlefish requires higher alkaline protease, α-amylase, and lipase activity to digest natural live food.

  14. 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

    abolish the protease inhibitory activity of PN-1. In the presence of the antibodies, PN-1 does not form a complex with its target proteases, but is recovered in a reactive centre cleaved form. Using site-directed mutagenesis, we mapped the three overlapping epitopes to an area spanning the gap between...

  15. Reduction of nitrobenzene with alkaline ascorbic acid: Kinetics and pathways

    International Nuclear Information System (INIS)

    Liang, Chenju; Lin, Ya-Ting; Shiu, Jia-Wei

    2016-01-01

    Highlights: • Alkaline ascorbic acid (a.k.a. vitamin C) is capable of reductively degrading NB. • The pH above the pK_a_2 of ascorbic acid increases reductive electron transfer to NB. • The rate equation for the reactions between NB and AA is determined. • NSB, AZOXY, and AZO are identified as intermediates and aniline as a final product. • Alkaline pH is essential for AA remediation of NB contaminated soils. - Abstract: Alkaline ascorbic acid (AA) exhibits the potential to reductively degrade nitrobenzene (NB), which is the simplest of the nitroaromatic compounds. The nitro group (NO_2"−) of NB has a +III oxidation state of the N atom and tends to gain electrons. The effect of alkaline pH ranging from 9 to 13 was initially assessed and the results demonstrated that the solution pH, when approaching or above the pK_a_2 of AA (11.79), would increase reductive electron transfer to NB. The rate equation for the reactions between NB and AA at pH 12 can be described as r = ((0.89 ± 0.11) × 10"−"4 mM"1"−"("a "+ "b") h"−"1) × [NB]"a "= "1"."3"5 "± "0"."1"0[AA]"b "= "0"."8"9 "± "0"."0"1. The GC/MS analytical method identified nitrosobenzene, azoxybenzene, and azobenzene as NB reduction intermediates, and aniline (AN) as a final product. These experimental results indicate that the alkaline AA reduction of NB to AN mainly proceeds via the direct route, consisting of a series of two-electron or four-electron transfers, and the condensation reaction plays a minor route. Preliminary evaluation of the remediation of spiked NB contaminated soils revealed that maintenance of alkaline pH and a higher water to soil ratio are essential for a successful alkaline AA application.

  16. Desorption of acetone from alkaline-earth exchanged Y zeolite after propane selective oxidation

    NARCIS (Netherlands)

    Xu, J.; Mojet, Barbara; van Ommen, J.G.; Lefferts, Leonardus

    2004-01-01

    The desorption of products from a series of alkaline-earth exchanged Y zeolites after room-temperature propane selective oxidation was investigated by in situ infrared and mass spectroscopy. The intermediate product, isopropylhydroperoxide (IHP), did not desorb during

  17. The dimer interfaces of protease and extra-protease domains influence the activation of protease and the specificity of GagPol cleavage.

    Science.gov (United States)

    Pettit, Steven C; Gulnik, Sergei; Everitt, Lori; Kaplan, Andrew H

    2003-01-01

    Activation of the human immunodeficiency virus type 1 (HIV-1) protease is an essential step in viral replication. As is the case for all retroviral proteases, enzyme activation requires the formation of protease homodimers. However, little is known about the mechanisms by which retroviral proteases become active within their precursors. Using an in vitro expression system, we have examined the determinants of activation efficiency and the order of cleavage site processing for the protease of HIV-1 within the full-length GagPol precursor. Following activation, initial cleavage occurs between the viral p2 and nucleocapsid proteins. This is followed by cleavage of a novel site located in the transframe domain. Mutational analysis of the dimer interface of the protease produced differential effects on activation and specificity. A subset of mutations produced enhanced cleavage at the amino terminus of the protease, suggesting that, in the wild-type precursor, cleavages that liberate the protease are a relatively late event. Replacement of the proline residue at position 1 of the protease dimer interface resulted in altered cleavage of distal sites and suggests that this residue functions as a cis-directed specificity determinant. In summary, our studies indicate that interactions within the protease dimer interface help determine the order of precursor cleavage and contribute to the formation of extended-protease intermediates. Assembly domains within GagPol outside the protease domain also influence enzyme activation.

  18. House dust mite major allergens Der p 1 and Der p 5 activate human airway-derived epithelial cells by protease-dependent and protease-independent mechanisms

    Directory of Open Access Journals (Sweden)

    Timmerman J André B

    2006-03-01

    Full Text Available Abstract House dust mite allergens (HDM cause bronchoconstriction in asthma patients and induce an inflammatory response in the lungs due to the release of cytokines, chemokines and additional mediators. The mechanism how HDM components achieve this is largely unknown. The objective of this study was to assess whether HDM components of Dermatophagoides pteronissinus with protease activity (Der p 1 and unknown enzymatic activity (Der p 2, Der p 5 induce biological responses in a human airway-derived epithelial cell line (A549, and if so, to elucidate the underlying mechanism(s of action. A549 cells were incubated with HDM extract, Der p 1, recombinant Der p 2 and recombinant Der p 5. Cell desquamation was assessed by microscopy. The proinflammatory cytokines, IL-6 and IL-8, were measured by ELISA. Intracellular Ca2+ levels were assessed in A549 cells and in mouse fibroblasts expressing the human protease activated receptor (PAR1, PAR2 or PAR4. HDM extract, Der p 1 and Der p 5 dose-dependently increased the production of IL-6 and IL-8. Added simultaneously, Der p 1 and Der p 5 further increased the production of IL-6 and IL-8. The action of Der p 1 was blocked by cysteine-protease inhibitors, while that of Der p 5 couldn't be blocked by either serine- or cysteine protease inhibitors. Der p 5 only induced cell shrinking, whereas HDM extract and Der p1 also induced cell desquamation. Der p 2 had no effect on A549 cells. Der p 1's protease activity causes desquamation and induced the release of IL6 and IL-8 by a mechanism independent of Ca2+ mobilisation and PAR activation. Der p 5 exerts a protease-independent activation of A549 that involves Ca2+ mobilisation and also leads to the production of these cytokines. Together, our data indicate that allergens present in HDM extracts can trigger protease-dependent and protease-independent signalling pathways in A549 cells.

  19. Serine protease inhibitors containing a Kunitz domain: their role in modulation of host inflammatory responses and parasite survival.

    Science.gov (United States)

    de Magalhães, Mariana T Q; Mambelli, Fábio S; Santos, Bruno P O; Morais, Suellen B; Oliveira, Sergio C

    2018-03-31

    Proteins containing a Kunitz domain have the typical serine protease inhibition function ranging from sea anemone to man. Protease inhibitors play major roles in infection, inflammation disorders and cancer. This review discusses the role of serine proteases containing a Kunitz domain in immunomodulation induced by helminth parasites. Helminth parasites are associated with protection from inflammatory conditions. Therefore, interest has raised whether worm parasites or their products hold potential as drugs for treatment of immunological disorders. Finally, we also propose the use of recombinant SmKI-1 from Schistosoma mansoni as a potential therapeutic molecule to treat inflammatory diseases. Copyright © 2018 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  20. PURIFICATION AND CHARACTERISATION OF ALKALINE ...

    African Journals Online (AJOL)

    Prof. Adipala Ekwamu

    There was no clear decrease in the yield seen in the bands and the loss of enzyme was not observed with the gel analysis. It may ... The native gel results show clear distinct bands for the 3 alkaline phosphotase isoenzymes ..... British Medical.