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Sample records for alkaline protease production

  1. Alkaline Protease Production by a Strain of Marine Yeasts

    Institute of Scientific and Technical Information of China (English)

    WANG Ping; CHI Zhenming; MA Chunling

    2006-01-01

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

  2. Alkaline protease production by a strain of marine yeasts

    Science.gov (United States)

    Ping, Wang; Zhenming, Chi; Chunling, Ma

    2006-07-01

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

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

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    Elham Dawoodi

    2014-12-01

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

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

    OpenAIRE

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

    2006-01-01

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

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

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    Mandana Lotfi

    2014-03-01

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

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

    Science.gov (United States)

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

    2015-01-01

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

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

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    Mohideen Askar Nawas P

    2015-07-01

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

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

    Institute of Scientific and Technical Information of China (English)

    2011-01-01

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

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

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    Nurullah Akcan

    2011-09-01

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

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

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    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.

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

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    Biswanath Bhunia

    2012-04-01

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

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

    OpenAIRE

    Adinarayana, Kunamneni; Jyothi, Bezawada; Ellaiah, Poluri

    2005-01-01

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

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

    OpenAIRE

    Ponnuswamy Vijayaraghavan; Sreekumar Saranya; Samuel Gnana Prakash Vincent

    2014-01-01

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

  14. 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.

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

    Science.gov (United States)

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

    2014-01-01

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

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

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    Mukundraj Govindrao Rathod

    2016-09-01

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

  17. 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.

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

    NARCIS (Netherlands)

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

    2004-01-01

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

  19. 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.

  20. 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.

  1. 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.

  2. 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++.

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

    Science.gov (United States)

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

    2013-09-01

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

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

    OpenAIRE

    Hamid Mukhtar; Ikramul Haq

    2013-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2009-01-01

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

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

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    Aruna K

    2014-09-01

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

  7. 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.

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

    Science.gov (United States)

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

    2014-05-01

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

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

    Science.gov (United States)

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

    2014-06-01

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

  10. 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.

  11. 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.

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

    Institute of Scientific and Technical Information of China (English)

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

    2005-01-01

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

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

    Science.gov (United States)

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

    2005-07-01

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

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

    Science.gov (United States)

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

    2014-10-03

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

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

    Science.gov (United States)

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

    2015-01-01

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

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

    Science.gov (United States)

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

    2002-12-01

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

  17. Stability and selectivity of alkaline proteases in hydrophilic solvents

    DEFF Research Database (Denmark)

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

    2008-01-01

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

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

    Science.gov (United States)

    Briki, Selmen; Hamdi, Olfa; Landoulsi, Ahmed

    2016-05-01

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

  19. OPTIMIZATION OF PROTEASE PRODUCTION FROM FUNGI ISOLATED FROM SOIL

    Directory of Open Access Journals (Sweden)

    Sonia Sethi

    2015-07-01

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

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

    Science.gov (United States)

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

    2015-02-01

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

  1. Purification and characterization of alkaline protease from Lysinibacillus fusiformis

    Directory of Open Access Journals (Sweden)

    Suppiah S*

    2012-08-01

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

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

    Science.gov (United States)

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

    2015-09-01

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

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

    Science.gov (United States)

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

    2011-01-01

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

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

    Science.gov (United States)

    Thangam, E Berla; Rajkumar, G Suseela

    2002-04-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2004-01-01

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

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

    Digital Repository Service at National Institute of Oceanography (India)

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

    and Chang CS. Bleach resistant alkaline protease produced by a Bacillus sp. Isolated from the Korean polychaete, Periserrula leucophryna. Proc Biochem 2004;39:1441-7. [12] Turkiewicz M, Gromek E, Kalinowska H and Zielinska M, Biosynthesis and properties...-stable, thiol-dependent serine alkaline protease from Bacillus mojavensis. Enzyme Microb Technol 2003;32:294-304. [21] Oh KH, Seong CS, Lee SW, Kwon OS and Park YS. Isolation of a psychrotrophic Azospirillum sp. and characterizaion of its extracellular...

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

    Directory of Open Access Journals (Sweden)

    Vijaya Bundela

    2013-03-01

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

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

    Science.gov (United States)

    Purohit, Megha K; Singh, Satya P

    2013-02-01

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

  9. Expression and characterization of Coprothermobacter proteolyticus alkaline serine protease

    Science.gov (United States)

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

  10. 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.

  11. 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.

  12. 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.

  13. 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.

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

    Institute of Scientific and Technical Information of China (English)

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

    2004-01-01

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

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

    Science.gov (United States)

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

    2016-05-01

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

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

    Science.gov (United States)

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

    2011-01-01

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

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

    Science.gov (United States)

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

    2016-05-01

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

  18. 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.

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

    Science.gov (United States)

    Gomaa, Eman Zakaria

    2013-01-01

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

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

    Science.gov (United States)

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

    2016-12-01

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

  1. 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

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

    Science.gov (United States)

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

    2012-06-01

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

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

    Science.gov (United States)

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

    2012-09-01

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

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

    Science.gov (United States)

    Shankar, Shiv; Laxman, Ryali Seeta

    2015-01-01

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

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

    Science.gov (United States)

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

    2012-01-01

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

  6. 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.

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

    Science.gov (United States)

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

    2012-02-03

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

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

    Directory of Open Access Journals (Sweden)

    Fikret Uyar

    2011-02-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2013-01-01

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

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

    Science.gov (United States)

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

    2013-11-01

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

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

    Science.gov (United States)

    Papagianni, M; Sergelidis, D

    2014-04-01

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

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

    Science.gov (United States)

    Gohel, S D; Singh, S P

    2015-01-01

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

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

    Science.gov (United States)

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

    2013-01-01

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

  14. 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...

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

    Institute of Scientific and Technical Information of China (English)

    马俊孝; 张赞剑; 史衍鲁; 卢彦梅; 刘伟

    2012-01-01

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

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

    Science.gov (United States)

    Niyonzima, Francois Niyongabo; More, Sunil

    2014-10-03

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

  17. 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.

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

    Science.gov (United States)

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

    2012-09-01

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

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

    DEFF Research Database (Denmark)

    Christiansen, Torben; Nielsen, Jens

    2002-01-01

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

  20. 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.

  1. 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.

  2. 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.

  3. Acid protease production in fungal root endophytes.

    Science.gov (United States)

    Mayerhofer, Michael S; Fraser, Erica; Kernaghan, Gavin

    2015-01-01

    Fungal endophytes are ubiquitous in healthy root tissue, but little is known about their ecosystem functions, including their ability to utilize organic nutrient sources such as proteins. Root-associated fungi may secrete proteases to access the carbon and mineral nutrients within proteins in the soil or in the cells of their plant host. We compared the protein utilization patterns of multiple isolates of the root endophytes Phialocephala fortinii s.l., Meliniomyces variabilis and Umbelopsis isabellina with those of two ectomycorrhizal (ECM) fungi, Hebeloma incarnatulum and Laccaria bicolor, and the wood-decay fungus Irpex lacteus at pH values of 2-9 on liquid BSA media. We also assessed protease activity using a fluorescently labeled casein assay and gelatin zymography and characterized proteases using specific protease inhibitors. I. lacteus and U. isabellina utilized protein efficiently, while the ECM fungi exhibited poor protein utilization. ECM fungi secreted metallo-proteases and had pH optima above 4, while other fungi produced aspartic proteases with lower pH optima. The ascomycetous root endophytes M. variabilis and P. fortinii exhibited intermediate levels of protein utilization and M. variabilis exhibited a very low pH optimum. Comparing proteolytic profiles between fungal root endophytes and fungi with well defined ecological roles provides insight into the ecology of these cryptic root associates.

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

    Science.gov (United States)

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

    1996-04-10

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

  5. 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.

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

    Science.gov (United States)

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

    2016-01-29

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

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

    Institute of Scientific and Technical Information of China (English)

    陈杰; 刘小宇; 焦炳华

    2013-01-01

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

  8. 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.

  9. 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

  10. 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.

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

    NARCIS (Netherlands)

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

    1992-01-01

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

  12. 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.

  13. 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...

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

    Science.gov (United States)

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

    2016-12-01

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

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

    Science.gov (United States)

    Huang, Rong; Yang, Qingjun; Feng, Hong

    2015-02-01

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

  16. 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.

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

    Science.gov (United States)

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

    2008-01-01

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

  18. Variation in Extracellular Protease Production among Clinical Isolates of Staphylococcus aureus Due to Different Levels of Expression of the Protease Repressor sarA

    OpenAIRE

    Karlsson, Anna; Arvidson, Staffan

    2002-01-01

    Staphylococcus aureus produces four major extracellular proteases: staphylococcal serine protease (V8 protease; SspA), cysteine protease (SspB), metalloprotease (aureolysin; Aur), and staphopain (Scp). Several in vitro studies have suggested that these enzymes are important virulence factors. Here we analyzed the protease production of 92 S. aureus strains from infected human soft tissue. Twenty-one strains produced variable zones of proteolysis on casein agar plates, while the remaining 71 s...

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

    Science.gov (United States)

    Johnston, David B; McAloon, Andrew J

    2014-02-01

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

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

    Science.gov (United States)

    Rajkumar, Renganathan; Kothilmozhian, Jayappriyan; Ramasamy, Rengasamy

    2011-06-01

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

  1. 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.)

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

    Science.gov (United States)

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

    2014-01-01

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

  3. Hydrogen production by alkaline water electrolysis

    OpenAIRE

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

    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...

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

    Directory of Open Access Journals (Sweden)

    Untung Susilo

    2016-11-01

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

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

    Institute of Scientific and Technical Information of China (English)

    黄继翔

    2011-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

    孙倩; 陈复生; 丁长河; 刘伯业

    2011-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Mital S. Dodia

    2006-09-01

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

  8. 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.

  9. Concomitant production of protease and lipase by Bacillus licheniformis VSG1: production, purification and characterization

    Directory of Open Access Journals (Sweden)

    R. Sangeetha

    2010-03-01

    Full Text Available This study was aimed at producing protease and lipase simultaneously on a common medium by Bacillus licheniformis VSG1, which was isolated from a tannery effluent. The effect of media composition with respect to protein source, lipid source and emulsifier on the production of protease and lipase was analysed. Both those enzymes were produced under optimized conditions like pH, temperature and incubation time. The enzyme mixture comprising of both protease and lipase was purified by ammonium sulphate precipitation, dialysis and gel filtration chromatography to obtain 20-fold pure enzymes. The purified enzyme mixture was characterized to determine the optimum pH and temperature of protease and lipase, the response of the enzymes to inhibitors, additives and solvents. The molecular weight of both the enzymes was determined as 40 kDa on SDS-PAGE. The concomitant production of protease and lipase and the purification of both the enzymes in a single mixture have industrial significance, as many industrial processes use both protease and lipase together.

  10. 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

    for product formation. The dynamics of the production of Savinuse(R) were studied during step changes in the dilution rate. During a step down in the dilution rate the specific production rate decreased immediately until it reached a new steady value. During a step-up an initial cease in the production rate...... was observed, but when glucose stopped to accumulate the production rate was regained. The glucose uptake was further investigated when chemostat cultures growing at different dilution rates were exposed to glucose pulses. The maximal glucose uptake capacity was found to be dependent on the initial specific......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...

  11. Optimum Production and Characterization of an Acid Protease from Marine Yeast Metschnikowia reukaufii W6b

    Institute of Scientific and Technical Information of China (English)

    LI Jing; PENG Ying; WANG Xianghong; CHI Zhenming

    2010-01-01

    The marine yeast strain W6b isolated from sediment of the South China Sea was found to produce a cell-bound acid protease.The crude acid protease produced by this marine yeast showed the highest activity at pH 3.5 and 40 ℃.The optimal pH and temperature for the crude acid protease were in agreement with those for acid protease produced by the terrestrial yeasts.The optimal medium of the acid protease production was seawater containing 1.0% glucose,1.5% casein,and 0.5% yeast extract,and the optimal cultivation conditions of the acid protease production were pH 4.0,a temperature of 25 ℃ and a shaking speed of 140 rmin-1.Under the optimal conditions,72.5 UmL-1 of acid protease activity could be obtained in cell suspension within 48 h of fermentation at shake flask level.The acid protease production was induced by high-molecular-weight nitrogen sources and repressed by low-molecular-weight nitrogen sources.Skimmed-milk-clotting test showed that the crude acid protease from the cell suspension of the yeast W6b had high skimmed milk coagulability.The acid protease produced by M.reukaufii W6b may have highly potential applications in cheese,food and fermentation industries.

  12. Optimum production and characterization of an acid protease from marine yeast Metschnikowia reukaufii W6b

    Science.gov (United States)

    Li, Jing; Peng, Ying; Wang, Xianghong; Chi, Zhenming

    2010-12-01

    The marine yeast strain W6b isolated from sediment of the South China Sea was found to produce a cell-bound acid protease. The crude acid protease produced by this marine yeast showed the highest activity at pH 3.5 and 40 °C. The optimal pH and temperature for the crude acid protease were in agreement with those for acid protease produced by the terrestrial yeasts. The optimal medium of the acid protease production was seawater containing 1.0% glucose, 1.5% casein, and 0.5% yeast extract, and the optimal cultivation conditions of the acid protease production were pH 4.0, a temperature of 25 °C and a shaking speed of 140 rmin-1. Under the optimal conditions, 72.5 UmL-1 of acid protease activity could be obtained in cell suspension within 48 h of fermentation at shake flask level. The acid protease production was induced by high-molecular-weight nitrogen sources and repressed by low-molecular-weight nitrogen sources. Skimmed-milk-clotting test showed that the crude acid protease from the cell suspension of the yeast W6b had high skimmed milk coagulability. The acid protease produced by M. reukaufii W6b may have highly potential applications in cheese, food and fermentation industries.

  13. 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.

  14. A Culture-Based Method for Determining the Production of Secreted Protease Inhibitors

    Science.gov (United States)

    Quintero, David; Bermudes, David

    2014-01-01

    We have developed a culture-based method for determining the production of secreted protease inhibitors. The assay utilizes standard proteolysis detection plates to support microbial growth followed by infiltrating the plate with a protease and subsequently detecting the remaining protein by trichloroacetic acid (TCA) precipitation, or by bromocreosol green (BCG) or Ponseau S (PS) staining. The presence of a protease inhibitor can be observed in the form of a protected zone of protein around the protease inhibitor-producing strain. Using the protease inhibitors α-2-macroglobulin, aprotinin, leupeptin, and bestatin and the primary and secondary forms of Photorhabdus luminescens in combination with the protease trypsin, we were able to demonstrate that the assay is specific for the cognate inhibitor of the protease and for bacteria secreting protease inhibitors. In addition, when casein-containing plates were used, the size of the diffusion zone was inversely correlated with the molecular weight of the inhibitor allowing a relative estimation of the protease inhibitor molecular weight. This assay is useful for detecting the presence of microbial secreted protease inhibitors and may reveal their production by microorganisms that were not previously recognized to produce them. PMID:24632514

  15. 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.

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

    Directory of Open Access Journals (Sweden)

    D. Kezia

    2011-03-01

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

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

    Institute of Scientific and Technical Information of China (English)

    许尨; 袁江兰; 靳艳; 邓川; 李文竹

    2012-01-01

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

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

    Science.gov (United States)

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

    2015-01-01

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

  19. Screening of protease-producing marine yeasts for production of the bioactive peptides

    Institute of Scientific and Technical Information of China (English)

    NI Xiumei; CHI Zhenming; LIU Zhiqiang; YUE Lixi

    2008-01-01

    Over 400 yeast strains from seawater and sediments were obtained,but only five strains named HN2-3,N13d,N13C,Mb5 and HN3-2 among them could form clear zones around their colonies on the double plates with 2.0% casein.Peptides in the hydroly-sate produced by the proteases from strains HN2-3 and N13d had higher angiotensin Ⅰ-converting-enzyme (ACE)-inhibitory ac-tivity.The two marine yeast strains were identified to be Aureobasidium pullulans according to the results of routine yeast identifi-cation and molecular methods.After purification of the proteases from the two marine yeast strains,it was found that the optimal pH for them was both 9.0,both of them were serine alkaline protease.However,the optimal temperature for the protease from the strain HN2-3 was 52℃ while that from strain N13d was 48℃.ACE-inhibitory activity of the peptides in the hydrolysate of shrimp protein produced by the purified protease from the strain HN2-3 was the highest while antioxidant activity in the hydroly-sate of spirulina protein produced by the purified protease from the strain N13d was the highest.

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

    Institute of Scientific and Technical Information of China (English)

    白英; 陈振涛

    2012-01-01

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

  1. 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.

  2. Alkaline Pretreatment of Sweet Sorghum Bagasse for Bioethanol Production

    Directory of Open Access Journals (Sweden)

    Yanni Sudiyani

    2016-08-01

    Full Text Available Lignocellulosic material, which consist mainly of cellulose, hemicelluloses and lignin, are among the most promising renewable feedstocks for the production of energy and chemicals.   The bagasse residue of sweet sorghum can be utilized as raw material for alternative energy such as bioethanol.  Bioethanol production consists of pretreatment, saccharification, fermentation and purification process.  The pretreatment process was of great importance to ethanol yield.  In the present study, alkaline pretreatment was conducted using a steam explosion reactor at 1300C with concentrations of NaOH  6, and 10% (kg/L for 10, and 30 min.  For ethanol production separated hydrolysis and fermentation (SHF and simultaneous saccharification and fermentation (SSF process were conducted with 30 FPU of Ctec2 and Htec2 enzyme and yeast of Saccharomyces cerevisiae.   The results showed that maximum cellulose conversion to total glucose plus xylose were showed greatest with NaOH 10% for 30 min.  The highest yield of ethanol is 96.26% and high concentration of ethanol 66.88 g/L were obtained at SSF condition during 48 h process. Using SSF process could increase yields and concentration of ethanol with less energy process. Article History: Received January 16th 2016; Received in revised form May 25th 2016; Accepted June 28th 2016; Available online How to Cite This Article: Sudiyani, Y., Triwahyuni, E., Muryanto, Burhani, D., Waluyo, J. Sulaswaty, A. and Abimanyu, H. (2016 Alkaline Pretreatment of Sweet Sorghum Bagasse for Bioethanol Production. Int. Journal of Renewable Energy Development, 5(2, 113-118. http://dx.doi.org/10.14710/ijred.5.2.113-118 

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

    Science.gov (United States)

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

    2011-01-01

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

  4. A novel extracellular protease from Pseudomonas aeruginosa MCM B-327: enzyme production and its partial characterization.

    Science.gov (United States)

    Zambare, Vasudeo; Nilegaonkar, Smita; Kanekar, Pradnya

    2011-02-28

    The focus of this study was on production, purification and characterization of dehairing protease from Pseudomonas aeruginosa MCM B-327, isolated from vermicompost pit soil. Optimum protease activity, 395 U mL(-1), was observed in the medium containing soybean meal and tryptone, at pH 7 and 30 °C. The crude enzyme exhibited dehairing activity. As compared to chemical method, enzymatic method of dehairing showed reduction in COD, TDS and TSS by 34.28%, 37.32% and 51.58%, respectively. Zymogram of crude enzyme on native-PAGE presented two bands with protease activity of molecular weights of 56 and 67 kDa. Both proteases showed dehairing activity. Out of these, 56kDa protease (PA02) was purified 3.05-folds with 2.71% recovery. The enzyme was active in pH range 7-9 and temperature 20-50 °C with optimum pH of 8 and temperature 35°C. Moreover, the enzyme activity of PA02 protease was not strongly inhibited by specific inhibitor showing the novel nature of enzyme compared to serine, cysteine, aspartyl and metalloproteases. Kinetic studies indicated that substrate specificity of PA02 protease was towards various natural and synthetic proteolytic substrates but inactive against collagen and keratin. These findings suggest protease secreted by P. aeruginosa MCM B-327 may have application in dehairing for environment-friendly leather processing.

  5. Statistical Optimization of Media Components for Production of Fibrinolytic Alkaline Metalloproteases from Xenorhabdus indica KB-3

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    Kumar Pranaw

    2014-01-01

    Full Text Available Xenorhabdus indica KB-3, a well-known protease producer, was isolated from its entomopathogenic nematode symbiont Steinernema thermophilum. Since medium constituents are critical to the protease production, the chemical components of the selected medium (soya casein digest broth were optimized by rotatable central composite design (RCCD using response surface methodology (RSM. The effects of all five chemical components (considered as independent variables, namely tryptone, soya peptone, dextrose, NaCl, and dipotassium phosphate, on protease production (dependent variable were studied, and it was found that tryptone and dextrose had maximum influence on protease production. The protease production was increased significantly by 66.31% under optimal medium conditions (tryptone—5.71, soya peptone—4.9, dextrose—1.45, NaCl—6.08, and dipotassium phosphate—0.47 in g/L. To best of knowledge, there are no reports on optimization of medium component for protease production by X. indica KB-3 using RSM and their application in fibrinolysis. This study will be useful for industrial processes for production of protease enzyme from X. indica KB-3 for its application in the field of agriculture and medicine.

  6. Production of Biodiesel Using Ethanol Way and Alkaline Catalyst

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    Cesar Aparecido da Silva

    2010-06-01

    Full Text Available The potential inputs to promote the supply of the demand for power generation has become the aim of several scientific researches to mitigate environmental impacts. The biodiesel is the highlight solution that can be obtained through the transesterification process. The aim this present work was the biodiesel production using ethanol and crude oil sunflower as inputs and potassium ethoxide such as catalyst for the rection. Were produced seven samples using different parameters. The product with high rate of ethyl ester was the one with catalyst and reaction time optimized. However, it has showed the presence of glycerol, suggesting the use of other unit operations such as cooling and centrifugation to improve the purity of the biodiesel formed is necessary. The parameters used in this experiment (oil, catalyst and water washing contents, reaction time, temperature and agitation speed showed critical endpoints to be monitored during the production of biodiesel due interfering the quality and yield to the final product. In addition, the inappropriate speed of agitation in the reactor for ethanol way in the presence of an alkaline catalyst can gelatinize the mixture of reactants due the emulsion formed.

  7. Production and Partial Characterization of an Alkaline Xylanase from a Novel Fungus Cladosporium oxysporum

    Directory of Open Access Journals (Sweden)

    Guo-Qiang Guan

    2016-01-01

    Full Text Available A new fungus Cladosporium oxysporum GQ-3 producing extracellular xylanase was isolated from decaying agricultural waste and identified based on the morphology and comparison of internal transcribed spacer (ITS rDNA gene sequence. C. oxysporum produced maximum xylanase activity of 55.92 U/mL with wheat bran as a substrate and NH4Cl as a nitrogen source. Mg2+ improved C. oxysporum xylanase production. Partially purified xylanase exhibited maximum activity at 50°C and pH 8.0, respectively, and showed the stable activity after 2-h treatment in pH 7.0–8.5 or below 55°C. Mg2+ enhanced the xylanase activity by 2% while Cu2+ had the highest inhibition ratio of 57.9%. Furthermore, C. oxysporum xylanase was resistant to most of tested neutral and alkaline proteases. Our findings indicated that Cladosporium oxysporum GQ-3 was a novel xylanase producer, which could be used in the textile processes or paper/feed industries.

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

    Science.gov (United States)

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

    2014-10-21

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

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

    Science.gov (United States)

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

    2016-07-01

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

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

    Science.gov (United States)

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

    2005-01-01

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

  11. Pyrosequencing reveals the key microorganisms involved in sludge alkaline fermentation for efficient short-chain fatty acids production.

    Science.gov (United States)

    Zheng, Xiong; Su, Yinglong; Li, Xiang; Xiao, Naidong; Wang, Dongbo; Chen, Yinguang

    2013-05-07

    Short-chain fatty acids (SCFAs) have been regarded as the excellent carbon source of wastewater biological nutrient removal, and sludge alkaline (pH 10) fermentation has been reported to achieve highly efficient SCFAs production. In this study, the underlying mechanisms for the improved SCFAs production at pH 10 were investigated by using 454 pyrosequencing and fluorescent in situ hybridization (FISH) to analyze the microbial community structures in sludge fermentation reactors. It was found that sludge fermentation at pH 10 increased the abundances of Pseudomonas sp. and Alcaligenes sp., which were able to excrete extracellular proteases and depolymerases, and thus enhanced the hydrolysis of insoluble sludge protein and polyhydroxyalkanoates (PHA). Meanwhile, the abundance of acid-producing bacteria (such as Clostridium sp.) in the reactor of pH 10 was also higher than that of uncontrolled pH, which benefited the acidification of soluble organic substrates. Further study indicated that sludge fermentation at pH 10 significantly decreased the number of methanogenic archaea, resulting in lower SCFAs consumption and lower methane production. Therefore, anaerobic sludge fermentation under alkaline conditions increased the abundances of bacteria involved in sludge hydrolysis and acidification, and decreased the abundance of methanogenic archaea, which favored the competition of bacteria over methanogens and resulted in the efficient production of SCFAs.

  12. Enhanced production and organic solvent stability of a protease fromBrevibacillus laterosporus strain PAP04

    Directory of Open Access Journals (Sweden)

    P. Anbu

    2016-01-01

    Full Text Available A bacterial strain (PAP04 isolated from cattle farm soil was shown to produce an extracellular, solvent-stable protease. Sequence analysis using 16S rRNA showed that this strain was highly homologous (99% to Brevibacillus laterosporus. Growth conditions that optimize protease production in this strain were determined as maltose (carbon source, skim milk (nitrogen source, pH 7.0, 40°C temperature, and 48 h incubation. Overall, conditions were optimized to yield a 5.91-fold higher production of protease compared to standard conditions. Furthermore, the stability of the enzyme in organic solvents was assessed by incubation for 2 weeks in solutions containing 50% concentration of various organic solvents. The enzyme retained activity in all tested solvents except ethanol; however, the protease activity was stimulated in benzene (74% followed by acetone (63% and chloroform (54.8%. In addition, the plate assay and zymography results also confirmed the stability of the PAP04 protease in various organic solvents. The organic solvent stability of this protease at high (50% concentrations of solvents makes it an alternative catalyst for peptide synthesis in non-aqueous media.

  13. Production and Characterization of Keratinolytic Protease from New Wool-Degrading Bacillus Species Isolated from Egyptian Ecosystem

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    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.

  14. Production and Characterization of Keratinolytic Protease from New Wool-Degrading Bacillus Species Isolated from Egyptian Ecosystem

    Science.gov (United States)

    Hassan, Mohamed A.; Haroun, Bakry M.; Amara, Amro A.; Serour, Ehab A.

    2013-01-01

    Novel keratin-degrading bacteria were isolated from sand soil samples collected from Minia Governorate, Egypt. In this study, the isolates were identified as Bacillus amyloliquefaciens MA20 and Bacillus subtilis MA21 based on morphological and biochemical characteristics as well as 16S rRNA gene sequencing. B. amyloliquefaciens MA20 and B. subtilis MA21 produced alkaline keratinolytic serine protease when cultivated in mineral medium containing 1% of wool straight off sheep as sole carbon and nitrogen source. The two strains were observed to degrade wool completely to powder at pH 7 and 37°C within 5 days. Under these conditions the maximum activity of proteases produced by B. amyloliquefaciens MA20 and B. subtilis MA21 was 922 and 814 U/ml, respectively. The proteases exhibited optimum temperature and pH at 60°C and 9, respectively. However, the keratinolytic proteases were stable in broad range of temperature and pH values towards casein Hammerstein. Furthermore the protease inhibitor studies indicated that the produced proteases belong to serine protease because of their sensitivity to PMSF while they were inhibited partially in presence of EDTA. The two proteases are stable in most of the used organic solvents and enhanced by metals suggesting their potential use in biotechnological applications such as wool industry. PMID:23936776

  15. A new tagged-TEV protease: construction, optimisation of production, purification and test activity.

    Science.gov (United States)

    Miladi, Baligh; Bouallagui, Hassib; Dridi, Cyrine; El Marjou, Ahmed; Boeuf, Guilhem; Di Martino, Patrick; Dufour, Florence; Elm'Selmi, Abdellatif

    2011-01-01

    The Tobacco Etch Virus (TEV) protease is frequently used in the cleavage of recombinant fusion proteins because of its efficiency and high specificity. In this work, we present a new recombinant form of TEV termed Streptag II-TEV for high-level production and purification of TEV protease from Escherichia coli and compare it to the hexahistidine (6xHis) tagged version of TEV. The effects of varying the host strain, the bacterial induction temperature (25, 30 and 37°C) and the IPTG inducer concentration on production and solubility of the two recombinant TEV proteases have been examined. Optimal Streptag II-TEV protein expression were obtained in the E. coli KRX strain under an induction temperature of 25°C in the presence of IPTG at 0.5 mM. In these conditions, soluble Streptag II-TEV and 6xHis-TEV proteases accounted for about 25% and 18% of total soluble proteins, respectively. About 70% of Streptag II-TEV and 60% of 6xHis-TEV were detected in the supernatant. Streptag II-TEV protease purifies to near homogeneity (approximately 99%) via a simple, single step Strep-Tactin chromatography purification protocol based on the presence of Streptag II. The higher production of Streptag II-TEV coupled to its purification and cleavage efficiencies make it an attractive alternate to 6xHis-TEV.

  16. Reprint of: A new tagged-TEV protease: Construction, optimisation of production, purification and test activity.

    Science.gov (United States)

    Miladi, Baligh; Bouallagui, Hassib; Dridi, Cyrine; El Marjou, Ahmed; Boeuf, Guilhem; Di Martino, Patrick; Dufour, Florence; Elm'selmi, Abdellatif

    2011-09-02

    The Tobacco Etch Virus (TEV) protease is frequently used in the cleavage of recombinant fusion proteins because of its efficiency and high specificity. In this work, we present a new recombinant form of TEV termed Streptag II-TEV for high-level production and purification of TEV protease from Escherichia coli and compare it to the hexahistidine (6xHis) tagged version of TEV. The effects of varying the host strain, the bacterial induction temperature (25, 30 and 37°C) and the IPTG inducer concentration on production and solubility of the two recombinant TEV proteases have been examined. Optimal Streptag II-TEV protein expression were obtained in the E. coli KRX strain under an induction temperature of 25°C in the presence of IPTG at 0.5mM. In these conditions, soluble Streptag II-TEV and 6xHis-TEV proteases accounted for about 25% and 18% of total soluble proteins, respectively. About 70% of Streptag II-TEV and 60% of 6xHis-TEV were detected in the supernatant. Streptag II-TEV protease purifies to near homogeneity (approximately 99%) via a simple, single step Strep-Tactin chromatography purification protocol based on the presence of Streptag II. The higher production of Streptag II-TEV coupled to its purification and cleavage efficiencies make it an attractive alternate to 6xHis-TEV.

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

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    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.

  18. Formation and regeneration of protoplasts for protease production in Streptomyces rimosus.

    Science.gov (United States)

    Yang, S S; Lei, C M

    2001-03-01

    To improve the formation and regeneration frequency of protoplasts for protease production, experiments were performed using a cultivation of Streptomyces rimosus TM-55 (CCRC 940061) in a Tryptic-soy broth (TSB) containing 2% of glycine for 2 days. It was found that the protoplast formation decreased with increased incubation temperature and increased ratio of culture broth to vessel volume. The optimal incubation temperature was 28 degreesC and the ratio of culture broth to vessel volume was 2:5. The hypertonic medium containing 10 mM MgCl2, 25 mM CaCl2 and 500 mM sucrose provided high stability for protoplasts. Supplementation with MgSO4, KCl and NaNO3 improved the regeneration frequency of protoplasts. The smear method had a higher protoplast regeneration frequency than the pour plate method. Protoplasts had protease productivity which was similar to that obtained with fresh mycelia, with each milliliter of culture broth yielding 141 units of protease with 3.5 x 10(8) protoplasts and 148 units of protease with 14.25 mg fresh mycelia respectively in a shaking culture, while the values were 15 and eight units of protease in a static culture.

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

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    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.

  20. 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. te

  1. Effects of culture conditions on ligninolytic enzymes and protease production by Phanerochaete chrysosporium in air

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    The production of ligninolytic enzymes and protease by Phanerochaete chrysosporium was investigated under different culture conditions. Different amounts of medium were employed in free and immobilized culture, together with two kinds of medium with different C/N ratios. Little lignin peroxidase (LiP) (< 2 U/L) was detected in free culture with nitrogen-limited medium (C/N ratio: 56/2.2 mmol/L), while manganese peroxidase (MnP) maximum activity was 231 and 240 U/L in 50 and 100 ml medium culture, respectively. Immobilized culture with 50 ml nitrogen-limited medium gave the highest MnP and LiP production with the maximum values of 410 and 721 U/L separately on day 5; however, flasks containing 100 ml nitrogen-limited medium only produced less MnP with a peak value of 290 U/L. Comparatively, carbon-limited medium (C/N ratio: 28/44 mmol/L) was adopted in culture but produced little MnP and LiP. Medium type had the greatest impact on protease production. Large amount of protease was produced due to glucose limitation. Culture type and medium volume influence protease activity corporately by affecting oxygen supply. The results implied shallow immobilized culture was a possible way to gain high production of ligninolytic enzymes.

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

    Science.gov (United States)

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

    2017-12-01

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

  3. Effect of carbon source on alkaline phosphatase production and excretion in Aspergillus caespitosus.

    Science.gov (United States)

    Guimarães, Luis Henrique Souza; Jorge, João Atilio; Terenzi, Héctor Francisco; Jamur, Maria Célia; Oliver, Constance; De Lourdes Teixeira De Moraes Polizeli, Maria

    2003-01-01

    The effect of several carbon sources on the production of alkaline phosphatase by the thermotolerant Aspergillus caespitosus was analysed. The fungus released high levels of alkaline phosphatases into the medium after being cultured for long periods with xylan or industrial residues such as wheat raw and sugar cane bagasse in the culture media. In contrast, the alkaline phosphatase activities were found only intracellulary when the fungus was cultured in glucose-supplemented media. The pH of the medium likely affects the process of enzyme secretion according to the carbon source used. Addition of xylan or industrial residues in the culture medium stimulated the secretion of phosphatases. In contrast, media supplemented with glucose or disaccharides promoted retention of these enzymes into the cells. The subcellular location activities of alkaline phosphatases were studied using histochemical and immunochemical methods and showed that alkaline phosphatases were present in the mycelial walls and septa.

  4. Increased performance of hydrogen production in microbial electrolysis cells under alkaline conditions.

    Science.gov (United States)

    Rago, Laura; Baeza, Juan A; Guisasola, Albert

    2016-06-01

    This work reports the first successful enrichment and operation of alkaline bioelectrochemical systems (microbial fuel cells, MFC, and microbial electrolysis cells, MEC). Alkaline (pH=9.3) bioelectrochemical hydrogen production presented better performance (+117%) compared to conventional neutral conditions (2.6 vs 1.2 litres of hydrogen gas per litre of reactor per day, LH2·L(-1)REACTOR·d(-1)). Pyrosequencing results of the anodic biofilm showed that while Geobacter was mainly detected under conventional neutral conditions, Geoalkalibacter sp. was highly detected in the alkaline MFC (21%) and MEC (48%). This is the first report of a high enrichment of Geoalkalibacter from an anaerobic mixed culture using alkaline conditions in an MEC. Moreover, Alkalibacter sp. was highly present in the anodic biofilm of the alkaline MFC (37%), which would indicate its potentiality as a new exoelectrogen.

  5. 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.

  6. Dairy products and the French paradox: Could alkaline phosphatases play a role?

    Science.gov (United States)

    Lallès, Jean-Paul

    2016-07-01

    The French paradox - high saturated fat consumption but low incidence of cardiovascular disease (CVD) and mortality - is still unresolved and continues to be a matter of debate and controversy. Recently, it was hypothesised that the high consumption of dairy products, and especially cheese by the French population might contribute to the explanation of the French paradox, in addition to the "(red) wine" hypothesis. Most notably this would involve milk bioactive peptides and biomolecules from cheese moulds. Here, we support the "dairy products" hypothesis further by proposing the "alkaline phosphatase" hypothesis. First, intestinal alkaline phosphatase (IAP), a potent endogenous anti-inflammatory enzyme, is directly stimulated by various components of milk (e.g. casein, calcium, lactose and even fat). This enzyme dephosphorylates and thus detoxifies pro-inflammatory microbial components like lipopolysaccharide, making them unable to trigger inflammatory responses and generate chronic low-grade inflammation leading to insulin resistance, glucose intolerance, type-2 diabetes, metabolic syndrome and obesity, known risk factors for CVD. Various vitamins present in high amounts in dairy products (e.g. vitamins A and D; methyl-donors: folate and vitamin B12), and also fermentation products such as butyrate and propionate found e.g. in cheese, all stimulate intestinal alkaline phosphatase. Second, moulded cheeses like Roquefort contain fungi producing an alkaline phosphatase. Third, milk itself contains a tissue nonspecific isoform of alkaline phosphatase that may function as IAP. Milk alkaline phosphatase is present in raw milk and dairy products increasingly consumed in France. It is deactivated by pasteurization but it can partially reactivate after thermal treatment. Experimental consolidation of the "alkaline phosphatase" hypothesis will require further work including: systematic alkaline phosphatase activity measurements in dairy products, live dairy ferments and

  7. Optimization of Culture Conditions and Inducers for Improved Protease Production by Penicillium griseofulvum LCJ231 under Submerged Fermentation

    Directory of Open Access Journals (Sweden)

    X. Josephine Jenitta

    2015-04-01

    Full Text Available The production of protease by Penicillium griseofulvum LCJ231 under submerged fermentation was studied with an objective to improve the production through medium optimization. Important nutritional and physical parameters were optimized for maximizing the protease production. The most suitable carbon source, nitrogen source and inducer for maximizing the protease production were studied. It was found that starch, yeast extract and casein were the suitable carbon source, nitrogen source and inducer respectively. The present study also explored the utilization of several agro-wastes as low-cost natural inducers for protease production. The addition of black gram husk as an inducer successfully enhanced the protease production (145.12 U/mL. Maximum production of the protease enzyme was found in the culture medium with initial medium pH of 8 and 2 g/L of inoculum. The results obtained in the present study demonstrate the potential use of the cheap and abundantly available black gram husk for the induction of proteases and thus offer a new approach for industrial enzyme production.

  8. Production, purification and characterisation of thermostable metallo-protease from newly isolated Bacillus sp. KG5

    Directory of Open Access Journals (Sweden)

    Nazenin Ahmetoglu

    2015-03-01

    Full Text Available Background: Due to the importance of microbial proteases in biotechnological applications, a number of microorganisms are being explored. The production, purification and characterisation of extracellular metallo-proteases by producing Bacillus sp. KG5 was studied. Material and Methods: Bacterial strain KG5 was isolated from Kos (Bingol hot spring. The strain KG5 was identified by morphological, physiological, biochemical and 16S rRNA gene sequencing. The effects of various parameters on protease production, such as time, temperature, pH, carbon and nitrogen sources and CaCl2 were studied. The enzyme was purified by ammonium sulphate precipitation and Sephadex G-75 gel permeation chromatography. Molecular weight was calculated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE and zymographic analysis. The effects of some metal ions, chelators and inhibitors on enzyme activity were determined. Results: The optimum temperature, pH and incubation period for protease production were 40-45°C, 7.0 and 24 h, respectively. It was determined that the best nitrogen sources were yeast extract and urea, while the best carbon sources were lactose and galactose. However, glucose as a source of carbon was found to inhibit the production of the enzyme. The maximum enzyme production was increased in the presence of CaCl2. The molecular weight of purified enzyme was found to be approximately 48 kDa. It was found that the enzyme was fully stable in the presence of 2 mM CaCl2 at 50°C after 120 min. Purified protease was significantly activated by Ca2+ and Mg2+, while it was greatly inhibited by Cu2+, Zn2+, Hg2+ and SDS as well as by the metal ion chelators ethylenediaminetetraacetic (EDTA and 1,10-phenanthroline. Phenylmethylsulfonyl fluoride (PMSF had a little effect on the enzyme. Conclusions: Our findings suggest the potential of this isolate for protease production and that this enzyme may be suitable for biotechnological applications.

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

    Institute of Scientific and Technical Information of China (English)

    柯野; 陈丹; 李家洲; 谢明权; 罗晓春

    2012-01-01

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

  10. Alkaline Ammonia Electrolysis on Electrodeposited Platinum for Controllable Hydrogen Production.

    Science.gov (United States)

    Gwak, Jieun; Choun, Myounghoon; Lee, Jaeyoung

    2016-02-19

    Ammonia is beginning to attract a great deal of attention as an alternative energy source carrier, because clean hydrogen can be produced through electrolytic processes without the emission of COx . In this study, we deposited various shapes of Pt catalysts under potentiostatic mode; the electrocatalytic oxidation behavior of ammonia using these catalysts was studied in alkaline media. The electrodeposited Pt was characterized by both qualitative and quantitative analysis. To discover the optimal structure and the effect of ammonia concentration, the bulk pH value, reaction temperature, and applied current of ammonia oxidation were investigated using potential sweep and galvanostatic methods. Finally, ammonia electrolysis was conducted using a zero-gap cell, producing highly pure hydrogen with an energy efficiency over 80 %.

  11. Enhancement of protease production by Pseudomonas aeruginosa isolated from dairy effluent sludge and determination of its fibrinolytic potential

    Institute of Scientific and Technical Information of China (English)

    Amrita Raj; Nancy Khess; Namrata Pujari; Sourav Bhattacharya; Arijit Das; Subbaramiah Sundara Rajan

    2012-01-01

    Objective: The present study aimed at isolating proteolytic bacteria from dairy effluent sludge, designing the process parameters for the enhanced production of protease and determination of its fibrinolytic potential. Methods: The dairy sludge was processed according to the microbiological criteria for the isolation of proteolytic bacteria. All the isolates were screened for their protease production ability and the isolate showing highest proteolysis was selected for further studies. Effects of various media components and process parameters like carbon and nitrogen supplementation, temperature, pH and incubation period were investigated. Partial purification of the protease was done using ammonium sulphate fractionation, following which its molecular weight and fibrinolytic activity were determined. Results: Based on the biochemical studies, the selected isolate was identified as Pseudomonas aeruginosa. The highest protease yield was obtained with maltose and yeast extract as supplements. The optimum pH, temperature and incubation period for protease production by the isolate was found to be 7.0, 37℃ and 48 h respectively. The partially purified enzyme preparation showed a single protein band in sodium dodecyl sulphate polyacrylamide gel electrophoresis, revealing the apparent molecular weight of the enzyme to be 35 kDa. The efficient removal of the blood stain emphasized its fibrinolytic potential. Conclusions: From the present study it is envisaged that cultural parameters significantly affect the protease production. Based upon the fibrinolytic activity, this protease may find broad applications in detergent and pharmaceutical industries.

  12. 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...

  13. Production of volatile fatty acids from sewage organic matter by combined bioflocculation and alkaline fermentation

    NARCIS (Netherlands)

    Khiewwijit, R.; Temmink, B.G.; Rijnaarts, H.H.M.; Keesman, K.J.

    2015-01-01

    This study explored the potential of volatile fatty acids (VFA) production from sewage by a combined high-loaded membrane bioreactor and sequencing batch fermenter. VFA production was optimized with respect to SRT and alkaline pH (pH 8–10). Application of pH shock to a value of 9 at the start of a s

  14. 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

    Animal manure digestion is associated with limited methane production, due to the high content in fibers, which are hardly degradable lignocellulosic compounds. In this study, different mechanical and thermal alkaline pretreatment methods were applied to partially degradable fibers, separated from...... 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......, enhancing fibers degradability by more than 4-fold. In continuous experiments, the thermal alkaline pretreatment, using 6% NaOH at 55 °C was proven to be the most efficient pretreatment method as the methane production was increased by 26%. The findings demonstrated that the methane production of the biogas...

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

    Science.gov (United States)

    Tsapekos, P; Kougias, Panagiotis G; Frison, A; Raga, R; Angelidaki, I

    2016-09-01

    Animal manure digestion is associated with limited methane production, due to the high content in fibers, which are hardly degradable lignocellulosic compounds. In this study, different mechanical and thermal alkaline pretreatment methods were applied to partially degradable fibers, separated from 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, enhancing fibers degradability by more than 4-fold. In continuous experiments, the thermal alkaline pretreatment, using 6% NaOH at 55°C was proven to be the most efficient pretreatment method as the methane production was increased by 26%. The findings demonstrated that the methane production of the biogas plants can be increased by further exploiting the fraction of the digested manure fibers which are discarded in the post-storage tank.

  16. 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.

  17. Enabling Low Cost Biopharmaceuticals: A Systematic Approach to Delete Proteases from a Well-Known Protein Production Host Trichoderma reesei.

    Directory of Open Access Journals (Sweden)

    Christopher P Landowski

    Full Text Available The filamentous fungus Trichoderma reesei has tremendous capability to secrete proteins. Therefore, it would be an excellent host for producing high levels of therapeutic proteins at low cost. Developing a filamentous fungus to produce sensitive therapeutic proteins requires that protease secretion is drastically reduced. We have identified 13 major secreted proteases that are related to degradation of therapeutic antibodies, interferon alpha 2b, and insulin like growth factor. The major proteases observed were aspartic, glutamic, subtilisin-like, and trypsin-like proteases. The seven most problematic proteases were sequentially removed from a strain to develop it for producing therapeutic proteins. After this the protease activity in the supernatant was dramatically reduced down to 4% of the original level based upon a casein substrate. When antibody was incubated in the six protease deletion strain supernatant, the heavy chain remained fully intact and no degradation products were observed. Interferon alpha 2b and insulin like growth factor were less stable in the same supernatant, but full length proteins remained when incubated overnight, in contrast to the original strain. As additional benefits, the multiple protease deletions have led to faster strain growth and higher levels of total protein in the culture supernatant.

  18. 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.

  19. Production of Amylases and Proteases by Bacillus caldolyticus from Food Industry Wastes

    Directory of Open Access Journals (Sweden)

    Thorsten Jamrath

    2012-01-01

    Full Text Available Amylases and proteases are utilized in industrial processes such as starch liquefaction or as supplements for washing agents. For these applications it is desirable to have enzymes active at high temperatures (>70 °C. In this work, thermostable α-amylase and neutral proteases were produced using the thermophilic strain Bacillus caldolyticus DSM 405. The goal of this work is to reduce the cost of production media by substituting expensive medium components such as prehydrolyzed starch and peptone, used in control fermentations, by inexpensive food industry wastes such as potato fruit water, potato pulp, cheese whey, draff, pea pulp, pea fruit water, bread residues, and pork blood. Comparative studies were conducted in shake flasks. With the use of such wastes, significant improvements in the activities of the enzyme α-amylase were obtained along with concomitant reductions in medium costs. With the use of pea pulp, 160 % increase in the activity of α-amylase was observed with 97 % reduction in medium costs compared to control medium. The cost of medium for the production of proteases also decreased by more than 50 %.

  20. Production of Alkaline Cellulase by Fungi Isolated from an Undisturbed Rain Forest of Peru

    Directory of Open Access Journals (Sweden)

    Karin Vega

    2012-01-01

    Full Text Available Alkaline cellulase producing fungi were isolated from soils of an undisturbed rain forest of Peru. The soil dilution plate method was used for the enumeration and isolation of fast growing cellulolytic fungi on an enriched selective medium. Eleven out of 50 different morphological colonies were finally selected by using the plate clearing assay with CMC as substrate at different pH values. All 11 strains produced cellulases in liquid culture with activities at alkaline pH values without an apparent decrease of them indicating that they are true alkaline cellulase producers. Aspergillus sp. LM-HP32, Penicillium sp. LM-HP33, and Penicillium sp. LM-HP37 were the best producers of FP cellulase (>3 U mL−1 with higher specific productivities (>30 U g−1 h−1. Three strains have been found suitable for developing processes for alkaline cellulase production. Soils from Amazonian rain forests are good sources of industrial fungi with particular characteristics. The results of the present study are of commercial and biological interest. Alkaline cellulases may be used in the polishing and washing of denim processing of the textile industry.

  1. Production of an extensive sunflower protein hydrolysate by sequential hydrolysis with endo- and exo-proteases.

    Directory of Open Access Journals (Sweden)

    Villanueva, Alvaro

    1999-12-01

    Full Text Available A high quality protein isolate has been obtained from defatted sunflower meal by alkaline extraction and isoelectric precipitation. Protein content was increased from 31.2 % in the defatted flour to 97 % in the protein isolate. The percentages of fiber, soluble sugars, polyphenols and residual lipids in the protein isolate were reduced to more than 90 % with respect to the defatted meal. The protein isolate was used as starting material for the generation of an extensive enzymatic protein hydrolysate. The hydrolysis was carried out in a pH stat using sequentially an endo-protease (Alcalase and an exo-protease (Flavourzyme. The protein hydrolysate, with a degree of hydrolysis of 50.7 %, was white and non bitter.

    Se ha obtenido un aislado proteico de alta calidad a partir de harina desengrasada de girasol, mediante extracción alcalina y precipitación isoeléctrica. Se incrementó el contenido proteico desde un 31.2 % en la harina desengrasada hasta un 97 % en el aislado proteico. Los porcentajes de fibra, azúcares solubles, polifenoles y lípidos residuales se redujeron en más del 90 % en el aislado proteico respecto a la harina desengrasada. Se usó el aislado proteico como material de partida para la producción de un hidrolizado enzimático proteico extenso. La hidrólisis se realizó en un reactor usando secuencialmente una endo-proteasa (Alcalasa y una exo-proteasa (Flavorzima. El hidrolizado proteico, con un grado de hidrólisis del 50.7 %, era blanco y no presentaba amargor.

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

    Science.gov (United States)

    Meevootisom, V; Niederpruem, D J

    1979-06-01

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

  3. Application of proteases as natural health products%蛋白酶的保健应用

    Institute of Scientific and Technical Information of China (English)

    夏建华

    2014-01-01

    Proteases are inseparable from our daily life. Their roles cover many aspects of cell cycles, me-tabolic regulations and body defenses. Actions of proteases consist of breaking down food proteins, assisting to decompose dying cells, neutralizing invading living agents or proteins and producing functional peptides which promote healings, to ensure a balanced cycling of metabolism and life. In the western countries, the history of using hydrolytic enzymes and protease included, as digestive aids could be traced back to at least 70 years ago. In contrast, the main stream of natural proteolytic enzymes which can be mass-produced in China has not yet been listed as ingredient for natural health products or dietary supplements. This paper briefly presents the characteristics, main functions, safety of application and the world market situation of several types of proteas-es. Application of proteases as natural health products seems to be promising in China.%蛋白酶与我们的日常生活密不可分,其作用涉及到细胞生命循环、代谢调节和身体防卫的许多方面,表现在:水解食物中的蛋白质,辅助分解衰竭细胞,清除入侵的微生物或蛋白质并产生可激发创伤恢复的功能性肽,以确保生命代谢循环的正常运行。西方国家利用包括蛋白酶在内的水解酶类保健至少有70年以上的历史,而我国尚未将一些主要的可大量生产的天然蛋白酶类列入天然保健品或膳食添加剂的原料名单中。本文简略介绍了几种主要蛋白酶的功能特性、应用安全性、国内外市场发展等方面的情况。蛋白酶作为天然保健品开发似有良好前景。

  4. Fuel ethanol production from alkaline peroxide pretreated corn stover

    Science.gov (United States)

    Corn stover (CS) has the potential to serve as an abundant low-cost feedstock for production of fuel ethanol. Due to heterogeneous complexity and recalcitrance of lignocellulosic feedstocks, pretreatment is required to break the lignin seal and/or disrupt the structure of crystalline cellulose to in...

  5. 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)

  6. 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; David Stewart; Bill Jones

    2005-10-01

    trends to subsequent alkaline-surfactant-polymer injected solution were observed. Aluminum citrate-polyacrylamide, resorcinol-formaldehyde, and the silicate-polyacrylamide gel systems did not produce significant incremental oil in linear corefloods. Both flowing and rigid flowing chromium acetate-polyacrylamide gels and the xanthan gum-chromium acetate gel system 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 the gels tested appeared to alter alkaline-surfactant-polymer solution oil recovery. Total waterflood plus chemical flood oil recovery sequence recoveries were all similar. Chromium acetate-polyacrylamide gel used to seal fractured core maintain fracture closure if followed by an alkaline-surfactant-polymer solution. Chromium acetate gels that were stable to injection of alkaline-surfactant-polymer solutions at 72 F were stable to injection of alkaline-surfactant-polymer solutions at 125 F and 175 F in linear corefloods. Chromium acetate-polyacrylamide gels maintained diversion capability after injection of an alkaline-surfactant-polymer solution in stacked; radial coreflood with a common well bore. Xanthan gum-chromium acetate gels maintained gel integrity in linear corefloods after injection of an alkaline-surfactant-polymer solution at 125 F. At 175 F, Xanthan gum-chromium acetate gels were not stable either with or without subsequent alkaline-surfactant-polymer solution injection. 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 when a gel was placed in the B sand. A sand and B sand alkaline

  7. 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.

  8. The effects of supplemental protease enzymes on production variables in lactating Holstein cows

    Directory of Open Access Journals (Sweden)

    Ekin Sucu

    2014-05-01

    Full Text Available A study was conducted to examine the effects of supplemental dietary protease enzymes on production variables in dairy cattle. Ninety-six multiparous lactating Holstein cows (624±62 kg body weight and 154±104 days in milk were blocked according to parity, days in milk, and previous milk production and randomly assigned to a control total mix ration (TMR or a TMR containing a blend of supplemental protease enzymes (PE; 4 g/cow/d in a crossover design with two 21-day experimental periods. Daily pen milk yield and dry matter intake (DMI were recorded and milk composition from all cows was determined on d 15, 17, 19 and 21 of each period. There was no treatment effect on milk yield (37.6 kg/d, but supplemental PE-fed cows consumed less DMI (P<0.05 compared to controls and therefore tended to have improved feed efficiency (P=0.06. Feeding supplemental PE decreased blood urea nitrogen (P<0.05 compared to the control cows. However, feeding PE had no effect on milk fat and protein content but tended (P=0.08 to increase milk lactose concentration and tended (P=0.10 to decrease milk urea nitrogen levels and somatic cell score. Results indicate that supplemental PE may enhance production efficiency and improve parameters of nitrogen status.

  9. Comparison of liquid hot water and alkaline pretreatments of giant reed for improved enzymatic digestibility and biogas energy production.

    Science.gov (United States)

    Jiang, Danping; Ge, Xumeng; Zhang, Quanguo; Li, Yebo

    2016-09-01

    Liquid hot water (LHW) and alkaline pretreatments of giant reed biomass were compared in terms of digestibility, methane production, and cost-benefit efficiency for electricity generation via anaerobic digestion with a combined heat and power system. Compared to LHW pretreatment, alkaline pretreatment retained more of the dry matter in giant reed biomass solids due to less severe conditions. Under their optimal conditions, LHW pretreatment (190°C, 15min) and alkaline pretreatment (20g/L of NaOH, 24h) improved glucose yield from giant reed by more than 2-fold, while only the alkaline pretreatment significantly (pAlkaline pretreatment achieved 27% higher net electrical energy production than that of non-pretreatment (3859kJ/kg initial total solids), but alkaline liquor reuse is needed for improved net benefit.

  10. Methane production and microbial community structure for alkaline pretreated waste activated sludge.

    Science.gov (United States)

    Sun, Rui; Xing, Defeng; Jia, Jianna; Zhou, Aijuan; Zhang, Lu; Ren, Nanqi

    2014-10-01

    Alkaline pretreatment was studied to analyze the influence on waste activated sludge (WAS) reduction, methane production and microbial community structure during anaerobic digestion. Methane production from alkaline pretreated sludge (A-WAS) (pH = 12) increased from 251.2 mL/Ld to 362.2 mL/Ld with the methane content of 68.7% compared to raw sludge (R-WAS). Sludge reduction had been improved, and volatile suspended solids (VSS) removal rate and protein reduction had increased by ∼ 10% and ∼ 35%, respectively. The bacterial and methanogenic communities were analyzed using 454 pyrosequencing and clone libraries of 16S rRNA gene. Remarkable shifts were observed in microbial community structures after alkaline pretreatment, especially for Archaea. The dominant methanogenic population changed from Methanosaeta for R-WAS to Methanosarcina for A-WAS. In addition to the enhancement of solubilization and hydrolysis of anaerobic digestion of WAS, alkaline pretreatment showed significant impacts on the enrichment and syntrophic interactions between microbial communities.

  11. Protease cell wall degradation of Chlorella vulgaris: effect on methane production.

    Science.gov (United States)

    Mahdy, Ahmed; Mendez, Lara; Blanco, Saul; Ballesteros, Mercedes; González-Fernández, Cristina

    2014-11-01

    In order to optimize the enzymatic dosage and microalgae biomass loads subjected to enzymatic hydrolysis prior anaerobic digestion of Chlorella vulgaris, organic matter solubilisation and methane production were investigated. Experimental data using protease dosage of 0.585 AU g DW(-1) showed that increasing biomass loads up to 65 g L(-1) did not affect markedly the hydrolysis efficiency (51%). Enzymatically pretreated biomasses subjected to anaerobic digestion enhanced methane production by 50-70%. The attempt of decreasing the enzymatic dosages revealed diminished hydrolysis efficiency concomitantly with a decreased methane production enhancement. In agreement with the good results observed for organic matter conversion into biogas, total nitrogen mineralization was attained for enzymatically pretreated biomass. Despite the high protein content of the biomass and the biocatalyst used in the present study no ammonia inhibition was detected.

  12. 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.

  13. Isolation and characterization of a newly isolated Pseudomonas mutant for protease production

    Directory of Open Access Journals (Sweden)

    Jayati Ray Dutta

    2006-01-01

    Full Text Available A potent bacterium for extracellular protease production was isolated from local soil and identified as Pseudomonas sp. RAJR 044. A mutant of this strain JNGR 242 with protease productivity 2.5 fold higher was obtained by ultraviolet irradiation under experimentally optimized conditions of pH 7.0, temperature of 34ºC, inoculum volume of 1.0 mL and incubation time of 24 hours. Comparative analysis of the chemical characteristics i.e. assimilation of carbon and nitrogen sources were also carried out. Maximum growth of the mutant strain in 2% gelatin agar plate was obtained in presence of dextrose (2%, maltose (2%, ammonium sulfate (2% and potassium nitrate (2% whereas, that of the parent strain was found in sucrose (2% and ammonium nitrate (2%. The purified proteases from both the strains (parent and mutant appeared as single homogeneous bands corresponding to 14.4 kDa molecular weight on SDS-PAGE. On studying the kinetic properties of both strains it was observed that the rate of casein hydrolysis was maximum at pH 8.0 and 7.0 and temperatures 45º C and 60º C for the parent and mutant strains respectively. It was also observed that both the extracellular proteases were inhibited by a serine protease inhibitor i.e. PMSF at 2mM concentration.Uma bactéria isolada do solo e identificada como Pseudomonas sp. RAJR 044 demonstrou ser uma potencial produtora de protéase extracelular. Um mutante JNGR 242 dessa espécie foi obtido mediante radiação ultravioleta sob condições experimentalmente otimizadas de pH 7,0, temperatura de 34ºC, volume de inóculo de 1,0 mL e tempo de incubação de 24 hora produziu 2,5 mais protease. Também foram realizadas análises comparativas das características química quanto assimilação de diferentes fontes de carbono e de nitrogênio. O crescimento máximo desse mutante na placa de ágar gelatina (2% foi obtido na presença de sacarose (2%, maltose (2%, sulfato de amônia (2% e nitrato de potássio (2%, ao

  14. Anaerobic digestion of poplar processing residues for methane production after alkaline treatment.

    Science.gov (United States)

    Yao, Yiqing; He, Mulan; Ren, Yubing; Ma, Liying; Luo, Yang; Sheng, Hongmei; Xiang, Yun; Zhang, Hua; Li, Qien; An, Lizhe

    2013-04-01

    Poplar processing residues were used for methane production by anaerobic digestion after alkaline treatment and methane production was measured. The highest methane production of 271.9 L/kg volatile solid (VS) was obtained at conditions of 35 g/L and 5.0% NaOH, which was 113.8% higher than non-alkaline treated samples, and 28.9% higher than that of corn straw, which is the conventional anaerobic digestion material in China. The maximal enhancement of 275.5% obtained at conditions of 50 g/L and 7.0% NaOH. Degradation of cellulose, hemicellulose and lignin after treatment increased by 4.0-9.0%, 3.3-6.2%, and 11.1-20.5%, respectively, with NaOH dose ranged from 3.0% to 7.0%. Scanning electron microscopy (SEM), FTIR spectra and Crystallinity measurements showed that the lignocellulosic structures were disrupted by NaOH. The results indicate poplar processing residues might be an efficient substrate for methane production after alkaline treatment.

  15. 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.

    , among which Chaetomium sp. (NIOCC 36) was found to grow in a wide range of pH (between 4 and 12). This alkaline tolerant fungus was further tested for production of alkaline cellulases (Beta-endoglucanase, Beta-exoglucanase, Beta-glucosidase) using...

  16. Production of xylanase and protease by Penicillium janthinellum CRC 87M-115 from different agricultural wastes.

    Science.gov (United States)

    Oliveira, Luciana A; Porto, Ana L F; Tambourgi, Elias B

    2006-04-01

    Five agricultural wastes were evaluated in submerged fermentation for xylanolytic enzymes production by Penicillium janthinellum. The wastes were hydrolyzed in acid medium and the liquid fraction was used for cultivation. Corn cob (55.3 U/mL) and oat husk (54.8 U/mL) were the best inducers of xylanase. Sugar cane bagasse (23.0 U/mL) and corn husk (23.8 U/mL) were moderately good, while cassava peel was negligible. Protease production was very low in all agro-industrial residues. The maximum biomass yields were 1.30 and 1.17 g/L for cassava peel and corn husk after 180 h, respectively. Xylanolytic activity showed a cell growth associated profile.

  17. Production of a protease inhibitor from edible mushroom Agaricus bisporus and its statistical optimization by response surface methodology.

    Science.gov (United States)

    Vishvakarma, Reena; Mishra, Abha

    2017-01-31

    The production of a protease inhibitor from Agaricus bisporus through solid state fermentation was studied. The purpose was to produce protease inhibitor from natural, cheap and readily available carbon and nitrogen sources. Solid state fermentation enhanced the mycelia growth and also gave a higher yield of the product. Further, fungal growth and other production parameters were statistically optimized. The specificity of the inhibitor was tested and was effective against trypsin. Screening of significant factors (wheat bran, cyanobacterial biomass, initial pH, temperature, incubation period, and moisture content and inoculum size) was done using Plackett-Burman Design. Central Composite Design was used to determine the optimized values of the significant variables which were found to be temperature (27.5 °C), incubation time (156 hrs.), cyanobacterial biomass (1 g) and moisture content (50%) and gave a statistical yield of 980 PIU/g which was 25.6% higher than experimental yield (780 PIU/g). The inhibitor was purified by ammonium sulphate precipitation and DEAE cellulose chromatography (yield 43.89% and 0.21% respectively) and subjected to Reversed-phase HPLC to validate its identity. Since protease inhibitors act against proteases, finding ample therapeutic roles; the isolated protease inhibitor from A. bisporus can also be a probable medicinal agent after its further characterization.

  18. Protease and urease production during utilization of diesel by fluorescent Pseudomonas species isolated from local soil

    Directory of Open Access Journals (Sweden)

    V.Umamaheswara Rao

    2009-12-01

    Full Text Available Background and objectives: Bacteria, most prevalently the Pseudomonas species possess high capacity to utilize and degrade"npetroleum hydrocarbons and are classified as the hydrocarbonoclastic microorganisms. Many species of the genus"nPseudomonas are notorious for their aerobic degradation capacity, extracellular enzyme production and are metabolically"nversatile organisms capable of utilizing a wide range of hydrocarbons and other compounds. In this study, the ability of diesel"nutilization by some locally isolated Pseudomonas species was tested."nMaterials and Methods: From a local red laterite soil, four different Pseudomonas species were isolated on King’s B medium,"ncharacterized, identified and tested their potential in utilizing diesel, a petroleum hydrocarbon. At the same time, production"nof protease and urease enzymes during the utilization of diesel was also assayed following the standard procedures."nResults: The isolates were grown well on diesel and subsequently produced the extracellular enzymes protease and urease"nat significant levels when compared to their production in the absence of diesel. Optimum temperature and pH for increased"ngrowth by four isolates was found to be 37oC and pH 8.0 indicating the maximum utilization of diesel. All the isolates showed"nmaximum growth in medium with 100% diesel than 100% glycerol as carbon source, when tested with different proportions"nof diesel and glycerol as carbon sources. Plasmid profile of the isolates revealed that, all four Pseudomonas isolates harbored"ntwo low molecular weight plasmids; one with 3 Kb size and the other with 10 kb to 12 Kb size."nConclusion: The four Pseudomonas isolates of the present study were found to have potential in diesel degradation and can"nbe recommended for bioremediation of sites that are contaminated with diesel.

  19. 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.

  20. Effect of thermal and alkaline pretreatment of giant miscanthus and Chinese fountaingrass on biogas production.

    Science.gov (United States)

    Nkemka, Valentine Nkongndem; Li, Yongqiang; Hao, Xiying

    2016-01-01

    Giant miscanthus (Miscanthus × giganteus) and Chinese fountaingrass (Pennisetum alopecuroides (L.) Spreng), cultivated for landscaping and soil conservation, are potential energy crops. The study investigated the effect of combined thermal and alkaline pretreatments on biogas production of these energy crops. The pretreatment included two types of alkali (6% CaO and 6% NaOH) at 22, 70 and 100 °C. The alkaline pretreatment resulted in a greater breakdown of the hemicellulose fraction, with CaO more effective than NaOH. Pretreatment of giant miscanthus with 6% CaO at 100 °C for 24 h produced a CH4 yield (313 mL g(-1) volatile solids (VS)) that was 1.7 times that of the untreated sample (186 mL g(-1) VS). However, pretreatment of Chinese fountaingrass with 6% CaO or 6% NaOH at 70 °C for 24 h resulted in similar CH4 yields (328 and 302 mL g(-1) VS for CaO and NaOH pretreatments) as the untreated sample (311 mL g(-1) VS). Chinese fountaingrass was more easily digestible but had a low overall CH4 yield per hectare (1,831 m(3) ha(-1) y(-1)) compared to giant miscanthus (6,868 m(3) ha(-1) y(-1)). This study demonstrates the potential of thermal/alkaline pretreatment and the use of giant miscanthus and Chinese fountaingrass for biogas production.

  1. Optimization of alkaline pretreatment of coffee pulp for production of bioethanol.

    Science.gov (United States)

    Menezes, Evandro G T; do Carmo, Juliana R; Alves, José Guilherme L F; Menezes, Aline G T; Guimarães, Isabela C; Queiroz, Fabiana; Pimenta, Carlos J

    2014-01-01

    The use of lignocellulosic raw materials in bioethanol production has been intensively investigated in recent years. However, for efficient conversion to ethanol, many pretreatment steps are required prior to hydrolysis and fermentation. Coffee stands out as the most important agricultural product in Brazil and wastes such as pulp and coffee husk are generated during the wet and dry processing to obtain green grains, respectively. This work focused on the optimization of alkaline pretreatment of coffee pulp with the aim of making its use in the alcoholic fermentation. A central composite rotatable design was used with three independent variables: sodium hydroxide and calcium hydroxide concentrations and alkaline pretreatment time, totaling 17 experiments. After alkaline pretreatment the concentration of cellulose, hemicellulose, and lignin remaining in the material, the subsequent hydrolysis of the cellulose component and its fermentation of substrate were evaluated. The results indicated that pretreatment using 4% (w/v) sodium hydroxide solution, with no calcium hydroxide, and 25 min treatment time gave the best results (69.18% cellulose remaining, 44.15% hemicelluloses remaining, 25.19% lignin remaining, 38.13 g/L of reducing sugars, and 27.02 g/L of glucose) and produced 13.66 g/L of ethanol with a yield of 0.4 g ethanol/g glucose.

  2. 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

    Alkaline wet oxidation pre-treatment (water, sodium carbonate, oxygen, high temperature and pressure) of wheat straw was performed as a 2(4-1) fractional factorial design with the process parameters: temperature, reaction time, sodium carbonate and oxygen. Alkaline wet oxidation was an efficient...... pre-treatment of wheat straw that resulted in solid fractions with high cellulose recovery (96%) and high enzymatic convertibility to glucose (67%). Carbonate and temperature were the most important factors for fractionation of wheat straw by wet oxidation. Optimal conditions were 10 min at 195...... the solid fraction (62%) did not provide a corresponding increase in the phenol monomer content but was correlated to high carboxylic acid concentrations. The degradation products in the hemicellulose fractions co-varied with the pre-treatment conditions in the principal component analysis according...

  3. Enhancing Production of Alkaline Polygalacturonate Lyase from Bacillus subtilis by Fed-Batch Fermentation

    OpenAIRE

    Mouyong Zou; Fenfen Guo; Xuezhi Li; Jian Zhao; Yinbo Qu

    2014-01-01

    Alkaline polygalacturonate lyase (PGL, EC 4.2.2.2) is an enzyme used in many industries. We developed a fed-batch fermentation process that combines the enzymatic pretreatment of the carbon source with controlling the pH of the fermentative broth to enhance the PGL production from Bacillus subtilis 7-3-3 to decrease the production cost. Maintaining the fermentation broth at pH 6.5 prior to feeding with ammonia and at pH 6.0 after feeding significantly improved PGL activity (743.5 U mL-1) comp...

  4. Optimization of cultural conditions for protease production by a fungal species

    Directory of Open Access Journals (Sweden)

    Kamath P

    2010-01-01

    Full Text Available Studies were carried out on a paddy soil fungal isolate identified to be a strain of Aspergillus niger from Manipal. The parameters that largely impact enzyme production viz., fermentation time, impeller speed, pH, temperature and nutrient supplements were studied. Optimization of production parameters for production of protease was done by the single-parameter mode. Casein served as substrate and proteolytic activity was estimated using Folin-Ciocalteau method at 660 nm. A maximum yield of 71.3 mg tyrosine/g casein substrate was produced in 96 h on a soluble starch medium at pH 4 in shake flask experiments. Production was carried out on a 3-liter fermenter and 40.7 mg of tyrosine was liberated/g of substrate. The enzyme was extracted with 50% ammonium sulfate and sodium dodecyl sulfate-Polyacrylamide gel electrophoresis showed two bands having mw 45.7 kDa and 38.5 kDa, respectively. The enzyme activity was found to be 147.84 U/ml.

  5. Integrated hydrometallurgical process for production of zinc from electric arc furnace dust in alkaline medium.

    Science.gov (United States)

    Youcai, Z; Stanforth, R

    2000-12-30

    In this study, a novel and integrated hydrometallurgical process for the production of zinc powder from electric arc furnace (EAF) dust in alkaline medium is reported. The dust is firstly hydrolysed in water, and then fused in caustic soda at 350 degrees C for 1h, followed by leaching in alkaline solution in which both zinc and lead are effectively extracted. Zinc powder is then produced by electrowinning from the leach solution after the lead is selectively removed by precipitation using sodium sulphide as precipitant. The EAF dust tested contained 25% Zn, 1.8% Pb and 33% Fe. It was found that 38% of zinc and 68% of lead could be extracted from the dust when leached directly in caustic soda solution. Leaching of zinc increased to 80% when dust was directly fused with caustic soda followed by alkaline leaching. However, the leaching further increased to 95% when the dust was hydrolysed first with water before fusion. Zinc powder with a purity of 99.95% was then produced by electrowinning from the lead depleted solution. Stainless electrodes were used as both anode and cathode.

  6. Electrocemical Production of Ocean Alkalinity for Carbon Dioxide and Acid Mitigation, and Hydrogen Generation

    Science.gov (United States)

    Rau, G. H.

    2008-12-01

    Various schemes have been proposed to increase air-to-sea CO2 transfer and storage, including the addition of alkalinity to the ocean. Examples include the addition of: Ca(OH)2 derived from the thermal calcination of limestone (Kheshgi, 1995), NaOH from the electrochemical splitting of salt (House et al., 2007), and CaCO3 to carbonate-undersaturated waters (Harvey, 2008). Diluted in the ocean (to pH1/2O2+H2+Ca(HCO3)2aq. Laboratory experiments showed that such a system can generate excess alkalinity and elevated pH in seawater that subsequently allowed the absorption of 0.8 mM atmospheric CO2. Thus at larger scales, wind-, wave-, or solar-powered, fixed/floating platforms at the shoreline, in coastal waters, or in the open ocean might be employed to electrochemically increase ocean alkalinity. Such platforms would then: 1) enhance the ocean's natural absorption of atmospheric CO2, 2) help neutralize or offset the effects of ongoing ocean acidification, via the calcium hydroxide and/or bicarbonate production, and 3) generate carbon-negative H2 in the ratio 22kg CO2 absorbed/kg H2 produced.

  7. Two-stage alkaline-enzymatic pretreatments to enhance biohydrogen production from sunflower stalks.

    Science.gov (United States)

    Monlau, Florian; Trably, Eric; Barakat, Abdellatif; Hamelin, Jérôme; Steyer, Jean-Philippe; Carrere, Hélène

    2013-01-01

    Because of their rich composition in carbohydrates, lignocellulosic residues represent an interesting source of biomass to produce biohydrogen by dark fermentation. Nevertheless, pretreatments should be applied to enhance the solubilization of holocelluloses and increase their further conversion into biohydrogen. The aim of this study was to investigate the effect of thermo-alkaline pretreatment alone and combined with enzymatic hydrolysis to enhance biohydrogen production from sunflower stalks. A low increase of hydrogen potentials from 2.3 ± 0.9 to 4.4 ± 2.6 and 20.6 ± 5.6 mL of H2 g(-1) of volatile solids (VS) was observed with raw sunflower stalks and after thermo-alkaline pretreatment at 55 °C, 24 h, and 4% NaOH and 170 °C, 1 h, and 4% NaOH, respectively. Enzymatic pretreatment alone showed an enhancement of the biohydrogen yields to 30.4 mL of H2 g(-1) of initial VS, whereas it led to 49 and 59.5 mL of H2 g(-1) of initial VS when combined with alkaline pretreatment at 55 and 170 °C, respectively. Interestingly, a diauxic effect was observed with sequential consumption of sugars by the mixed cultures during dark fermentation. Glucose was first consumed, and once glucose was completely exhausted, xylose was used by the microorganisms, mainly related to Clostridium species.

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

    Science.gov (United States)

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

    2013-03-01

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

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

    Science.gov (United States)

    Lane, Michael D; Seelig, Burckhard

    2016-05-01

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

  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. Comparative performance of enzymatic and combined alkaline-enzymatic pretreatments on methane production from ensiled sorghum forage.

    Science.gov (United States)

    Rollini, Manuela; Sambusiti, Cecilia; Musatti, Alida; Ficara, Elena; Retinò, Isabella; Malpei, Francesca

    2014-12-01

    This study investigated the effect of enzymatic and combined alkaline-enzymatic pretreatments on chemical composition and methane production from ensiled sorghum forage. Four commercial enzymatic preparations were tested and the two yielding the highest sugars release were added to evaluate any hydrolytic effect on both untreated and alkaline pretreated samples. In the combined alkaline-enzymatic pretreatment trials, the highest sugar release was found with Primafast and BGL preparations (added at a final concentration 0.12 and 0.20 mL/g TS, respectively), with a total monomeric content of 12 and 6.5 g/L. Fibre composition analysis confirmed that the combined alkaline-enzymatic pretreatment led to cellulose (up to 32 %) and hemicelluloses (up to 56 %) solubilisation, compared to the enzymatic pretreatment alone. BMP tests were performed on both untreated and pretreated samples, and time courses of methane production were fitted. Both enzymatic and combined alkaline-enzymatic pretreatment led to a methane production increase (304 and 362 mL CH4/g VS), compared to that of untreated sorghum (265 mL CH4/g VS), as  +15 and  +37 %, respectively. Moreover, higher specific methane production rates, compared to that of untreated sorghum (20.31 mL CH4/g VS/d), were obtained by applying the enzymatic and combined alkaline-enzymatic pretreatment (33.94 and 31.65 mL CH4/g VS/d), respectively.

  12. ONO 3403, a synthetic serine protease inhibitor, inhibits lipopolysaccharide-induced tumor necrosis factor-alpha and nitric oxide production and protects mice from lethal endotoxic shock

    NARCIS (Netherlands)

    Tumurkhuu, Gantsetseg; Koide, Naoki; Hiwasa, Takaki; Ookoshi, Motohiro; Dagvadorj, Jargalsaikhan; Noman, Abu Shadat Mohammod; Iftakhar-E-Khuda, Imtiaz; Naiki, Yoshikazu; Komatsu, Takayuki; Yoshida, Tomoaki; Yokochi, Takashi

    2011-01-01

    ONO 3403, a new synthetic serine protease inhibitor, is a derivative of camostat mesilate and has a higher protease-inhibitory activity. The effect of ONO 3403 on lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF)-alpha and nitric oxide (NO) production in RAW 264.7 macrophage-like cells wa

  13. A new condensation product for zinc plating from non-cyanide alkaline bath

    Indian Academy of Sciences (India)

    Y Arthoba Naik; T V Venkatesha

    2005-08-01

    Zinc electroplating from non-cyanide alkaline solution is carried out in the presence of condensation product formed between DL-alanine (DLA) and glutaraldehyde. The bath constituents and bath variables are optimized through standard Hull cell experiments. The current efficiency and the throwing power are measured. High shift of potential towards more cathodic direction was observed in presence of addition agents. Corrosion resistance test reveals good protection of base metal by zinc coating obtained from the developed electrolyte. SEM photomicrographs show fine-grained deposit in the presence of condensation product. IR spectrum of the scraped deposit shows the inclusion of the condensation product in the deposit during plating. The consumption of brightener in the lab-scale is 6 mLL-1 for 1000 amp-hour.

  14. 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

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

    Institute of Scientific and Technical Information of China (English)

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

    2012-01-01

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

  16. Contemporaneous Production of Amylase and Protease through CCD Response Surface Methodology by Newly Isolated Bacillus megaterium Strain B69

    Directory of Open Access Journals (Sweden)

    Rajshree Saxena

    2014-01-01

    Full Text Available The enormous increase in world population has resulted in generation of million tons of agricultural wastes. Biotechnological process for production of green chemicals, namely, enzymes, provides the best utilization of these otherwise unutilized wastes. The present study elaborates concomitant production of protease and amylase in solid state fermentation (SSF by a newly isolated Bacillus megaterium B69, using agroindustrial wastes. Two-level statistical model employing Plackett-Burman and response surface methodology was designed for optimization of various physicochemical conditions affecting the production of two enzymes concomitantly. The studies revealed that the new strain concomitantly produced 1242 U/g of protease and 1666.6 U/g of amylase by best utilizing mustard oilseed cake as the substrate at 20% substrate concentration and 45% moisture content after 84 h of incubation. An increase of 2.95- and 2.04-fold from basal media was observed in protease and amylase production, respectively. ANOVA of both the design models showed high accuracy of the polynomial model with significant similarities between the predicted and the observed results. The model stood accurate at the bench level validation, suggesting that the design model could be used for multienzyme production at mass scale.

  17. Alkaline direct transesterification of different species of Stichococcus for bio-oil production.

    Science.gov (United States)

    Gargano, Immacolata; Marotta, Raffaele; Andreozzi, Roberto; Olivieri, Giuseppe; Marzocchella, Antonio; Spasiano, Danilo; Pinto, Gabriele; Pollio, Antonino

    2016-12-25

    The cost of bio-oil refining from microalgal biomass can be significantly reduced by combining extraction and transesterification. The characterisation and optimisation of the combined steps have been carried out on strains of Stichococcus bacillaris, focusing on catalyst type and concentration, reaction time and temperature, methanol/biomass ratio, pre-mixing time and water content in the biomass. The bio-oil yield has been referenced as production of fatty acid methyl esters (FAMEs). The maximum yield (∼17%) was achieved using dried biomass with alkaline catalyst at 60°C and methanol/biomass weight ratio of 79:1. Alkaline catalyst conditions gave faster reaction rates and higher bio-oil yields than acid catalyst. Yield was also strongly affected by water content in the biomass. A mechanistic interpretation has been proposed to elucidate the effect of the different operating conditions. However, the structural characteristics of the Chlorophyta cell wall can be very different, leading to different bio-oil yields when the same protocol is applied. Therefore, the optimised protocol of direct transesterification for Stichococcus bacillaris strains was tested on other Stichococcus strains and several other Chlorophyta species characterised by a different cell wall structure. It was clearly demonstrated that different results for bio-oil yield were obtained within the same microalgal species and much more within different microalgal genera.

  18. 75 FR 80826 - Compliance Policy Guide Sec. 527.300 Dairy Products-Microbial Contaminants and Alkaline...

    Science.gov (United States)

    2010-12-23

    ... HUMAN SERVICES Food and Drug Administration Compliance Policy Guide Sec. 527.300 Dairy Products... Compliance Policy Guide Sec. 527.300 Dairy Products-- Microbial Contaminants and Alkaline Phosphatase... other indicators of inadequate pasteurization or post-pasteurization contamination of dairy...

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

    Institute of Scientific and Technical Information of China (English)

    翟爱华; 李新华

    2012-01-01

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

  20. Evaluation of electricity production from alkaline pretreated sludge using two-chamber microbial fuel cell.

    Science.gov (United States)

    Xiao, Benyi; Yang, Fang; Liu, Junxin

    2013-06-15

    Electricity production from alkaline pretreated sludge was evaluated using a two-chamber microbial fuel cell (MFC). The electricity production was found to be stable over a long period of time (approximately 17 d) with voltage outputs and power densities of 0.47-0.52 V and 46.80-55.88 mW/m(2), respectively. The anode resistance was the main internal resistance (73.2%) of MFC in the stable stage. Most soluble organic matters (proteins and carbohydrates) in the anode chamber were first degraded and converted into volatile fatty acids (0-15 d), which were then degraded and converted into electricity and methane (15-29 d). The insoluble organics were solubilized thereby decreasing the sludge concentration and reducing the sludge mass. Methane was produced in the anode chamber owing to the growth of methanogens, which did not obviously affect the electricity production. The change in humic-like substances displayed a positive correlation with the electricity production of the MFC. Microbial analysis showed that methanogens and electricity-producing bacteria co-existed mostly on the surface as well as inside the anode. Decreasing the anode resistance and increasing the anode utilization could enhance the electricity production.

  1. 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.

  2. Simultaneous determination of sulpiride and its alkaline degradation product by second derivative synchronous fluorescence spectroscopy.

    Science.gov (United States)

    Abdelal, Amina; El-Enany, Nahed; Belal, Fathalla

    2009-12-15

    Simple and sensitive synchronous fluorimetric, and second derivative synchronous fluorometric methods were developed for the validated and simultaneous determination of sulpiride (SLP) and its alkaline degradation product (DSLP). The method is based on measuring the synchronous fluorescence of both the drug and its degradation product in borate buffer of pH 8 at Deltalambda of 45 nm. The peak amplitude ((2)D) was measured at 295.5 and 342 nm for SLP and DSLP, respectively. The different experimental parameters affecting the synchronous fluorescence intensity of both compounds were studied and optimized. The fluorescence-concentration plots were rectilinear over the range of 0.05-1.0 and 2-10 microg mL(-1) for SLP and DSLP, respectively. The limits of detection (LOD) were 0.02 and 0.4 microg mL(-1) and quantification limits (LOQs) were 0.05 and 1.2 microg mL(-1) for SLP and DSLP, respectively. The proposed methods were successfully applied to commercial capsules and tablets. Statistical comparison of the results with those of the official method revealed good agreement and proved that there were no significant difference in the accuracy and precision between the two methods, respectively. The method was utilized to study the kinetics of the alkaline induced degradation of the drug. The application was further extended to include the in vivo and in vitro determination of sulpiride. The mean % recoveries (n=3) were 100.22+/-2.04 and 92.00+/-3.00 for spiked and real human plasma, respectively.

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

    Science.gov (United States)

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

    2013-02-01

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

  4. Presence of a lethal protease in the extracellular products of Vibrio splendidus-Vibrio lentus related strains.

    Science.gov (United States)

    Farto, R; Armada, S P; Montes, M; Perez, M J; Nieto, T P

    2006-12-01

    The presence of a lethal extracellular 39-kDa protease, a virulence determinant of a Listonella pelagia strain which produces vibriosis in turbot, was determined in the extracellular products (ECP) of 33 Vibrionaceae strains. Both immunological and enzymatic techniques distinguished this specific protease from other Vibrionaceae proteins. It was detected in 15% (5/33) of the ECPs assayed belonging to strains of the Vibrio splendidus-V. lentus related group isolated in Galician aquaculture systems (NW Spain). As these strains were associated with diseased octopus and cultured turbot, were able to colonize the internal organs of fish and produced a lethal ECP for fish, they are a potential risk for the health of reared aquatic organisms.

  5. Volatile fatty acids production from anaerobic treatment of cassava waste water: effect of temperature and alkalinity.

    Science.gov (United States)

    Hasan, Salah Din Mahmud; Giongo, Citieli; Fiorese, Mônica Lady; Gomes, Simone Damasceno; Ferrari, Tatiane Caroline; Savoldi, Tarcio Enrico

    2015-01-01

    The production of volatile fatty acids (VFAs), intermediates in the anaerobic degradation process of organic matter from waste water, was evaluated in this work. A batch reactor was used to investigate the effect of temperature, and alkalinity in the production of VFAs, from the fermentation of industrial cassava waste water. Peak production of total volatile fatty acids (TVFAs) was observed in the first two days of acidogenesis. A central composite design was performed, and the highest yield (3400 mg L(-1) of TVFA) was obtained with 30°C and 3 g L(-1) of sodium bicarbonate. The peak of VFA was in 45 h (pH 5.9) with a predominance of acetic (63%) and butyric acid (22%), followed by propionic acid (12%). Decreases in amounts of cyanide (12.9%) and chemical oxygen demand (21.6%) were observed, in addition to the production of biogas (0.53 cm(3) h(-1)). The process was validated experimentally and 3400 g L(-1) of TVFA were obtained with a low relative standard deviation.

  6. 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.

  7. 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

  8. 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

  9. Fungal protease.

    NARCIS (Netherlands)

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

    1994-01-01

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

  10. Application of Protease in the Processing of Aquatic Product%水产品加工中蛋白酶的应用研究

    Institute of Scientific and Technical Information of China (English)

    李咏梅

    2011-01-01

    蛋白酶是催化肽键水解的一群酶类,在水产品加工中应用广泛。对低值水产品以及水产副产品的综合加工中蛋白酶的应用进行了初步探讨,分析了如何更有效地开发利用蛋白酶资源。%Protease is a group of enzymes to catalyze the cleavage of peptide bonds,and nowadays protease is widely applied on aquatic product processing. The application of protease in the multipurpose processing of low value aquatic product and aquatic by-product was introduced,furthermore how to effectively develop and utilize protease resource was analyzed.

  11. Invited review: The application of alkaline phosphatase assays for the validation of milk product pasteurization.

    Science.gov (United States)

    Rankin, S A; Christiansen, A; Lee, W; Banavara, D S; Lopez-Hernandez, A

    2010-12-01

    Standard practices for indirectly assessing the pasteurization status of milk products are primarily based on the thermal inactivation kinetics of the endogenous milk enzyme, alkaline phosphatase (ALP). This assessment provides an invaluable, if not required, tool for both regulatory and in-house process control and validation. Endogenous milk ALP manifests a slightly higher heat resistance than the pathogenic microflora upon which pasteurization time and temperature requirements are based. Hence, ALP activity is recognized and accepted as the method of choice for the rapid validation of milk product pasteurization. However, ALP assays have notable limitations that must be understood if they are to be administered and interpreted correctly and the results are to be applied judiciously. Issues such as the reactivation of heat-denatured ALP and the presence of both heat-stable and -labile microbial ALP are addressed. A discussion of ALP in the milk of nonbovine species is presented based on the limited literature available. Some discussion of research involving alternative pasteurization indicators also is presented. This article is intended to summarize the pertinent details of the ALP assay for dairy products (noting the basis and limitations of various methods) and the processing, handling, and known compositional factors that influence the assay results.

  12. ALKALINE PRETREATMENT OF SPRUCE AND BIRCH TO IMPROVE BIOETHANOL AND BIOGAS PRODUCTION

    Directory of Open Access Journals (Sweden)

    Azam Jeihanipour

    2010-05-01

    Full Text Available Alkaline pretreatment with NaOH under mild operating conditions was used to improve ethanol and biogas production from softwood spruce and hardwood birch. The pretreatments were carried out at different temperatures between minus 15 and 100ºC with 7.0% w/w NaOH solution for 2 h. The pretreated materials were then enzymatically hydrolyzed and subsequently fermented to ethanol or anaerobically digested to biogas. In general, the pretreatment was more successful for both ethanol and biogas production from the hardwood birch than the softwood spruce. The pretreatment resulted in significant reduction of hemicellulose and the crystallinity of cellulose, which might be responsible for improved enzymatic hydrolyses of birch from 6.9% to 82.3% and spruce from 14.1% to 35.7%. These results were obtained with pretreatment at 100°C for birch and 5°C for spruce. Subsequently, the best ethanol yield obtained was 0.08 g/g of the spruce while pretreated at 100°C, and 0.17 g/g of the birch treated at 100°C. On the other hand, digestion of untreated birch and spruce resulted in methane yields of 250 and 30 l/kg VS of the wood species, respectively. The pretreatment of the wood species at the best conditions for enzymatic hydrolysis resulted in 83% and 74% improvement in methane production from birch and spruce.

  13. Inhibition of Plasmodium falciparum oocyst production by membrane-permeant cysteine protease inhibitor E64d.

    NARCIS (Netherlands)

    Eksi, S.; Czesny, B.; Gemert, G.J.A. van; Sauerwein, R.W.; Eling, W.M.C.; Williamson, K.C.

    2007-01-01

    During asexual intraerythrocytic growth, Plasmodium falciparum utilizes hemoglobin obtained from the host red blood cell (RBC) as a nutrient source. Papain-like cysteine proteases, falcipains 2 and 3, have been reported to be involved in hemoglobin digestion and are targets of current antimalarial d

  14. Processing Proteases

    DEFF Research Database (Denmark)

    Ødum, Anders Sebastian Rosenkrans

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

  15. The production of glucose from corn stalk using hydrothermal process with pre-treatment ultrasound assisted alkaline

    Science.gov (United States)

    Yolanda, Dora; Prasutiyo, Indry; Trisanti, P. N.; Sumarno

    2015-12-01

    The production of glucose from corn stalk by using subcritical hydrothermal technology is studied in this work. Ultrasound-assisted alkaline delignification methods are used as pre-treatment. The corn stalk powder were pretreated with ultrasound-assisted alkaline (NaOH 2% w/w, solid to liquid ratio 1:22 w/v) at room temperature and 30 minutes. After pre-treatment, solid residue and liquid fractions are separated by filtration. Pretreated solids are further submitted to hydrothermal process for glucose production. Hydrothermal process was carried out at 100 Bar and 120°C in various times. The solid product was characterized by SEM and XRD. And liquid product was analysis using DNS method to determine percentage of glucose. From XRD analysis showed that crystallinity of material was lower than delignification product.

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

    Directory of Open Access Journals (Sweden)

    Anthonia Odiase

    2012-09-01

    Full Text Available   Background. 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. Results. 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

  18. 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...

  19. Studies on the production of alkaline α-amylase from Bacillus subtilis CB-18

    Directory of Open Access Journals (Sweden)

    Ogbonnaya Nwokoro

    2015-03-01

    Full Text Available Background. Amylases are among the main enzymes used in food and other industries. They hydrolyse starch molecules into polymers composing glucose units. Amylases have potential applications in a number of industrial processes including foods and pharmaceutical industries. Alkaline α-amylase has the potential of hydrolysing starch under alkaline pH and is useful in the starch and textile industries and as an ingredient of detergents. Amylases are produced from plants, however, microbial production processes have dominated applications in the industries. Optimization of microbial production processes can result in improved enzyme yields. Material and methods. Amylase activity was assayed by incubating the enzyme solution (0.5 ml with 1% soluble starch (0.5 ml in 0.1 M Tris/HCl buffer (pH 8.5. After 30 minutes, the reaction was stopped by the addition of 4 mL of 3,5-dinitrosalicylic acid (DNS reagent then heated for 10 min in boiling water bath and cooled in a refrigerator. Absorbance readings were used to estimate the units of enzyme activity from glucose standard curve. Hydrolysed native starches from cassava, rice, corn, coco yam, maize and potato and soluble starch were adjusted to pH 8.5 prior to incubation with crude enzyme solution. Reducing sugars produced were therefore determined. The effect of pH on enzyme activity of the alkaline α-amylase was determined by using buffer solutions of different pH (potassium phosphate buffer, 6.0–7.0; Tris-HCl buffer 7.5 to 9.0 and carbonate/bicarbonate buffer, pH 9.5–11 for enzyme assay. The pH stability profi le of the enzyme was determined by incubating 0.5 ml of α-amylase enzyme in 0.1 M Tris/HCl buffer (pH 8.5 and 0.5 ml of 1% (w/v soluble starch (Merck in 0.1 M Tris/HCl buffer (pH 8.5 for 3 h in various buffers. The effect of temperature on enzyme activity was studied by incubating 0.5 mL of the enzyme solution contained in the test tube and 0.5 mL of 1% soluble starch (Merck solution

  20. 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

  1. 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.

  2. 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.

  3. Optimizing production of hydroxyapatite from alkaline residue for removal of Pb2+ from wastewater

    Science.gov (United States)

    Yan, Yubo; Wang, YanPeng; Sun, Xiuyun; Li, Jiansheng; Shen, Jinyou; Han, Weiqing; Liu, Xiaodong; Wang, Lianjun

    2014-10-01

    Alkaline residue, a common solid waste generated from the ammonia-soda process for the production of soda ash, has been converted into hydroxyapatite for Pb2+ 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 Pb2+ 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 Pb2+ 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 Pb2+ 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 Pb2+ from contaminated soil.

  4. 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.

  5. 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.

  6. Bioethanol Production from Coconut Fiber Using Alkaline Pretreatment and Acid Hydrolysis Method

    Directory of Open Access Journals (Sweden)

    Asyeni Miftahul Jannah

    2015-01-01

    Full Text Available Supporting Indonesia government program to decrease fuel consumption, using renewable energy such of bioethanol is one of the best ways. This research was done in order to utilize agriculture waste (coconut fiber as raw material to produce bioetanol. However, coconut fiber contents lignin that will inhibit conversion process of glucose into ethanol. In this research, pretreatment steps aim to release and breakdown lignin in coconut fiber. Pretreatment was conducted by using alkaline method with 3% Sodium Hydroxide solution. Hydrolysis method was used to produce glucose by using Sulfuric Acid solution with various concentrations (1%, 2%, 3%, and 4 % while in fermentation process used Saccharomyces cerevisiae with various times (5, 7, 9, and 11 days and distillation used to get pure product of bioethanol. The results showed that higher H2SO4 concentration using on hydrolysis process made more glucose converted to bioethanol. The highest bioethanol content produced was 5.9420% from sample of 4% H2SO4 in 7 days of fermentation.

  7. Acetone-butanol-ethanol production from corn stover pretreated by alkaline twin-screw extrusion pretreatment.

    Science.gov (United States)

    Zhang, Yuedong; Hou, Tongang; Li, Bin; Liu, Chao; Mu, Xindong; Wang, Haisong

    2014-05-01

    In this study, the alkaline twin-screw extrusion pretreated corn stover was subjected to enzymatic hydrolysis after washing. The impact of solid loading and enzyme dose on enzymatic hydrolysis was investigated. It was found that 68.2 g/L of total fermentable sugar could be obtained after enzymatic hydrolysis with the solid loading of 10 %, while the highest sugar recovery of 91.07 % was achieved when the solid loading was 2 % with the cellulase dose of 24 FPU/g substrate. Subsequently, the hydrolyzate was fermented by Clostridium acetobutylicum ATCC 824. The acetone-butanol-ethanol (ABE) production of the hydrolyzate was compared with the glucose, xylose and simulated hydrolyzate medium which have the same reducing sugar concentration. It was shown that 7.1 g/L butanol and 11.2 g/L ABE could be produced after 72 h fermentation for the hydrolyzate obtained from enzymatic hydrolysis with 6 % solid loading. This is comparable to the glucose and simulated hydrozate medium, and the overall ABE yield could reach 0.112 g/g raw corn stover.

  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. 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. 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

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

    Institute of Scientific and Technical Information of China (English)

    徐雅芫; 李吕木; 许发芝; 丁小玲; 钱坤

    2011-01-01

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

  12. Enhanced Production of Extracellular Alkaline Lipase by an Improved Strain of Pseudomonas aeruginosa MTCC 10,055

    Directory of Open Access Journals (Sweden)

    Deepali Bisht

    2012-01-01

    Full Text Available Problem statement: Lipases are industrially important enzymes having applications in numerous industries. For easy commercialization it is necessary to produce lipases at industrial level which could be achieved by strain improvement and medium formulation. Approach: In the present study strain improvement of Pseudomonas aeruginosa MTCC 10,055 was done by chemical mutagenesis using mutagen 4-nitroquinoline1-oxide for alkaline lipase production. Different fermentation parameters affecting lipase production were optimized using one-variable-at-a-time approach. Results: The selected mutant (M-05 exhibited 3.6-fold higher productivity over wild type. Maximum alkaline lipase was produced when culture was incubated at 35°C with initial medium pH 9.0 in 28 h with inoculum density 0.5% (v/v (Abs610-1.0. Supplementation of production medium with combination of castor oil and starch as carbon source and Triton-X-100 as surfactant significantly influenced the alkaline lipase production. The composition of fully optimized medium was determined to be (g L-1: (NH42SO4, 1.0; KH2PO4, 0.6; MgSO4, 0.4; yeast extract, 0.2; castor oil, 2.0; starch 20.0; gum arabic, 5.0; Triton-X-100, 1.0. An overall 14-fold enhanced production was achieved after complete medium optimization. Conclusion/Recommendations: The improved strain was capable to produce high titer of alkaline lipase at flask level, which can be examined at fermentor level to obtain sufficient enzyme yield to meet the world wide industrial demand.

  13. Protease-dependent activation of epithelial cells by fungal allergens leads to morphologic changes and cytokine production

    NARCIS (Netherlands)

    Kauffman, HF; Tomee, JFC; van de Riet, MA; Timmerman, AJB; Borger, P

    2000-01-01

    Background: Proteases in extracts of Aspergillus fumigatus cause epithelial cell desquamation and release of proinflammatory cytokines. Objective: We sought to assess protease activity in Alternaria alternata, Cladosporium herbarum, and Aspergillus fumigatus extracts and study the ability of these e

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

    OpenAIRE

    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 ...

  15. Protease inhibitor

    DEFF Research Database (Denmark)

    2009-01-01

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

  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. 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.

  18. 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

  19. 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

  20. Exceptional enhancement of H2 production in alkaline environment over plasmonic Au/TiO2 photocatalyst under visible light

    Directory of Open Access Journals (Sweden)

    Xianguang Meng

    2015-10-01

    Full Text Available A reaction environment modulation strategy was employed to promote the H2 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 H2 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 H2 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.

  1. 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.

  2. Recent progress in alkaline direct ethylene glycol fuel cells for sustainable energy production

    Science.gov (United States)

    An, L.; Chen, R.

    2016-10-01

    Alkaline direct ethylene glycol fuel cells are one of the most promising power sources for portable, mobile and stationary power applications, primarily because this type of fuel cell runs on a sustainable fuel and the key materials that constitute the fuel cell are relatively inexpensive. This review article summarizes and discusses the past investigations on the development of alkaline direct ethylene glycol fuel cells, including the physical and chemical processes through the fuel cell structure, the electrocatalytic oxidation and electrocatalysts of ethylene glycol, the singe-cell performance, and innovative system designs.

  3. Extracellular protease derived from lactic acid bacteria stimulates the fermentative lactic acid production from the by-products of rice as a biomass refinery function.

    Science.gov (United States)

    Watanabe, Masanori; Techapun, Charin; Kuntiya, Ampin; Leksawasdi, Noppol; Seesuriyachan, Phisit; Chaiyaso, Thanongsak; Takenaka, Shinji; Maeda, Isamu; Koyama, Masahiro; Nakamura, Kozo

    2017-02-01

    A lactic acid producing bacterium, Lactobacillus rhamnosus M-23, newly isolated from a rice washing drainage storage tank was found to produce l-(+)-lactic acid from a non-sterilized mixture of rice washing drainage and rice bran without any additions of nutrients under the simultaneous saccharification and fermentation (SSF) process. This strain has the ability to utilize the non-sterilized rice washing drainage and rice bran as a source of carbohydrate, saccharifying enzymes and nutrients for lactic acid production. Observation of extracellular protease activity in SSF culture broth showed that a higher protease activity was present in strain M-23 than in other isolated lactic acid producing bacteria (LABs). To investigate the structural changes of solid particles of rice washing drainage throughout LAB cultivation, scanning electron microscopic (SEM) observation and Fourier transform infrared-spectroscopy (FT-IR) analysis were performed. The results of the SEM observation showed that the surface material could be removed from solid particles of rice washing drainage treated by culture broth (supernatant) of strain M-23, thus exposing the crystal structure of the starch particle surface. The results of the FT-IR analysis revealed that the specific transmittance decrease of the CC and CO stretching and OH group of the solid particles of the rice washing drainage were highly correlated with the produced lactic acid concentration and extracellular protease activity, respectively. These results demonstrate the high lactic acid producing ability of strain M-23 from a non-sterilized mixture of rice washing drainage and rice bran under the SSF condition due to the removal of proteinaceous material and exposure of the starch particle surface by extracellular protease.

  4. saeRS and sarA act synergistically to repress protease production and promote biofilm formation in Staphylococcus aureus.

    Directory of Open Access Journals (Sweden)

    Lara N Mrak

    Full Text Available Mutation of the staphylococcal accessory regulator (sarA limits biofilm formation in diverse strains of Staphylococcus aureus, but there are exceptions. One of these is the commonly studied strain Newman. This strain has two defects of potential relevance, the first being mutations that preclude anchoring of the fibronectin-binding proteins FnbA and FnbB to the cell wall, and the second being a point mutation in saeS that results in constitutive activation of the saePQRS regulatory system. We repaired these defects to determine whether either plays a role in biofilm formation and, if so, whether this could account for the reduced impact of sarA in Newman. Restoration of surface-anchored FnbA enhanced biofilm formation, but mutation of sarA in this fnbA-positive strain increased rather than decreased biofilm formation. Mutation of sarA in an saeS-repaired derivative of Newman (P18L or a Newman saeRS mutant (ΔsaeRS resulted in a biofilm-deficient phenotype like that observed in clinical isolates, even in the absence of surface-anchored FnbA. These phenotypes were correlated with increased production of extracellular proteases and decreased accumulation of FnbA and/or Spa in the P18L and ΔsaeRS sarA mutants by comparison to the Newman sarA mutant. The reduced accumulation of Spa was reversed by mutation of the gene encoding aureolysin, while the reduced accumulation of FnbA was reversed by mutation of the sspABC operon. These results demonstrate that saeRS and sarA act synergistically to repress the production of extracellular proteases that would otherwise limit accumulation of critical proteins that contribute to biofilm formation, with constitutive activation of saeRS limiting protease production, even in a sarA mutant, to a degree that can be correlated with increased enhanced capacity to form a biofilm. Although it remains unclear whether these effects are mediated directly or indirectly, studies done with an sspA::lux reporter suggest they

  5. Production of proteases from industrial wastes through solid-state fermentation at different scales. Potential applications

    OpenAIRE

    2014-01-01

    En aquest treball es proposa un procés biotecnològic respectuós del medi ambient per reduir l'impacte negatiu de l'augment dels residus industrials, a causa de l'acceleració del creixement de la població mundial en les últimes dècades. Consisteix en la valorització de residus locals riques en nitrogen, com la fibra de soja, els residus de pel i closca de cafè, per fermentació en estat sòlid (SSF) per obtenir un catalitzador biològic, com ara proteases. Es van dur a terme experiments de SSF en...

  6. CHARACTERIZATION OF PROTEASE PRODUCED BY “JINHUA”FUNGI FROM FUZHUAN BRICK TEA%茯砖茶中“金花”菌产蛋白酶特性的研究

    Institute of Scientific and Technical Information of China (English)

    丁婷; 吕嘉枥; 侯蓓

    2011-01-01

    在不同培养时间段对“金花”菌产酸、中、碱性蛋白酶酶活进行测定,研究了不同碳源、氮源、无机盐等培养条件对产中性及碱性蛋白酶酶活的影响.结果表明:“金花”菌所产蛋白酶以中性和碱性蛋白酶为主,最适碳源为乳糖,氮源为麸皮,最适无机盐为氯化钠.在此条件下 “金花”菌产中性蛋白酶的酶活可达到237 U/g,产碱性蛋白酶的酶活可达236 U/g.“金花”菌对茯砖茶的消化作用有促进作用.%The acid protease activity, neutral protease and alkaline protease which produced by "Jinhua" fungi was measured at different cultural time. The effect of addition of carbon sources, nitrogen sources and inorganic salts on producing neutral and alkaline protease was studied. The results show that the many produced protease of "Jinhua" fungi was neutral protease and alkaline protease. Carbon source and nitrogen source preferred for protease production were lactose and bran, while inorganic salts preferred were sodium chloride. Under these conditions, the neutral protease activity was 237 U/g and the alkaline protease activity was 236 U/g.

  7. Alkaline peroxide pretreatment of corn stover for enzymatic saccharification and ethanol production

    Science.gov (United States)

    Alkaline hydrogen peroxide (AHP) pretreatment and enzymatic saccharification were evaluated for conversion of corn stover cellulose and hemicellulose to fermentable sugars. Corn stover used in this study contained 37.0±0.2% cellulose, 26.8±0.2% hemicellulose and 18.0±0.1% lignin on dry basis. Unde...

  8. Neutralization/prevention of acid rock drainage using mixtures of alkaline by-products and sulfidic mine wastes.

    Science.gov (United States)

    Alakangas, Lena; Andersson, Elin; Mueller, Seth

    2013-11-01

    Backfilling of open pit with sulfidic waste rock followed by inundation is a common method for reducing sulfide oxidation after mine closure. This approach can be complemented by mixing the waste rock with alkaline materials from pulp and steel mills to increase the system's neutralization potential. Leachates from 1 m3 tanks containing sulfide-rich (ca.30 wt %) waste rock formed under dry and water saturated conditions under laboratory conditions were characterized and compared to those formed from mixtures. The waste rock leachate produced an acidic leachate (pH9). The decrease of elemental concentration in the leachate was most pronounced for Pb and Zn, while Al and S were relatively high. Overall, the results obtained were promising and suggest that alkaline by-products could be useful additives for minimizing ARD formation.

  9. 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)

  10. 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.

  11. Improved production of protease-resistant phytase by Aspergillus oryzae and its applicability in the hydrolysis of insoluble phytates.

    Science.gov (United States)

    Sapna; Singh, Bijender

    2013-08-01

    Among three hundred isolates of filamentous fungi, Aspergillus oryzae SBS50 secreted higher phytase activity at pH 5.0, 35 °C and 200 rpm after 96 h of fermentation. Starch and beef extract supported the highest phytase production than other carbon and nitrogen sources. A nine-fold improvement in phytase production was achieved due to optimization. Supplementation of the medium with inorganic phosphate repressed the enzyme synthesis. Among surfactants tested, Tween 80 increased fungal growth and phytase production, which further resulted in 5.4-fold enhancement in phytase production. The phytase activity was not much affected by proteases treatment. The enzyme resulted in the efficient hydrolysis of insoluble phytate complexes (metal- and protein-phytates) in a time dependent manner. Furthermore, the hydrolysis of insoluble phytates was also supported by scanning electron microscopy. The enzyme, being resistant to trypsin and pepsin, and able to hydrolyze insoluble phytates, can find an application in the animal food/feed industry for improving nutritional quality and also in combating environmental phosphorus pollution and plant growth promotion.

  12. Medium initial pH and carbon source stimulate differential alkaline cellulase time course production in Stachybotrys microspora.

    Science.gov (United States)

    Ben Hmad, Ines; Abdeljalil, Salma; Saibi, Walid; Amouri, Bahia; Gargouri, Ali

    2014-03-01

    The production profile of cellulases of the mutant strain A19 from the filamentous fungus Stachybotrys microspora was studied in the presence of various carbon sources (glucose, lactose, cellulose, carboxymethylcellulose (CMC), and wheat bran) and a range of medium initial pH (5, 7, and 8). Two extracellular cellulases from the Stachybotrys strain (endoglucanases and β-glucosidases) were monitored by enzymatic assay, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and zymogram analysis. Glucose and lactose repressed CMCase time course production while they permitted a strong β-glucosidase one. On Avicel cellulose, CMC, and wheat bran, both activities were highly produced. Wheat bran (WB) is the best carbon source with an optimum of production at days 5 and 6. The production kinetics of both activities were shown to depend on the medium initial pH, with a preference for neutral or alkaline pH in the majority of conditions. The exception concerned the β-glucosidase which was much more produced at acidic pH, on glucose and cellulose. Most interestingly, a constitutive and conditional expression of an alkaline endoglucanase was revealed on the glucose-based medium at an initial pH of 8 units. The zymogram analysis confirmed such conclusions and highlighted that carbon sources and the pH of the culture medium directed a differential induction of various endoglucanases and β-glucosidases.

  13. 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.

  14. 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

  15. Searching for halo-alkalophilic proteases maintaining stability and activity in hydrophilic aprotic solvents as biocatalysts in carbohydrate chemistry

    DEFF Research Database (Denmark)

    Pedersen, Lars Haastrup; Mørkholt, Camilla Kær; Nielsen, Carsten Bue

    2012-01-01

    and other carbohydrates (1, 2). Therefore, we are searching for new bacterial halo-alkaline proteases from extreme environments in Denmark. So far we have identified a number of interesting isolates showing proteolytic activity at pH 7 and 10. Whole genome Illumina Hiseq 2000 amplicon sequencing, de novo...... assembly of the genome and automatic annotation of genes in the genome was carried out by RAST to find the genes responsible for protease production. The amino-acid sequence of the expressed proteolytic proteins was analyzed using tandem MS. The isolates identified so far belong to Bacillus sp. One...... of the interesting genomes with a total length of 4.1 Mb was assembled in 42 contigs with a total of 3070 known genes of which 39 are encoding for proteases (one serine endopeptidase). Another genome was assembled in 219 contigs with 4004 coding sequences and 2821 known genes of which 36 are encoding for proteases...

  16. 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

    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 str...

  17. Methods for producing dairy products, in particular cheese using lactic acid bacteria provided with additional neutral protease activity

    NARCIS (Netherlands)

    Mierau, Igor; van den Burg, Lambertus; Kok, Jan; Venema, Gerhardus

    1997-01-01

    The present invention relates to a method for carrying out a process of biotransformation of a substrate whereby at least one lactic acid bacterium comprising a gene encoding a neutral protease having an altered resulting activity, e.g. a neutral protease having an altered stability and/or specifici

  18. 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.

  19. 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.

  20. Alkalinity and structure of soils determine the truffle production in the Pyrenean Regions

    Directory of Open Access Journals (Sweden)

    Benoit Jaillard

    2014-08-01

    Full Text Available Aim of study: The program "Typology of truffle stations in the Pyrenean Regions" aimed to define the ecological conditions and culture practices that favor Tuber melanosporum growth and fruiting in this area.Area of study: Navarra, Catalonia, Midi-Pyrénées and Languedoc-Roussillon.Material and methods: The program was based on the survey of 212 wild and cultivated truffle beds of evergreen oaks (Quercus ilex. The data collected in the field consisted of photographs, samples of soil, roots and mycorrhizae, and information on cultural practices followed by truffle growers.Main results: (i truffle soils are alkaline, from neutral, dolomitic, to moderately or very calcareous soils; (ii truffle soils are light, well-structured and stable to water immersion; (iii mycelium that colonizes roots survives in suboptimal conditions, but it does not necessarily bear ascocarps. Finally our results suggest that T. melanosporum is a relatively ubiquitous fungus able to grow, or at least to persist, in a wide range of physical and chemical soil conditions. We propose a probabilistic model of the environment favorable for fruiting, built around a two-dimensional graph with an axis for the chemical conditions, like soil alkalinity, and another axis for the physical conditions, like soil structure. Research highlights: Soil alkalinity and structure allow to built a convenient representation of the ecological capacity of a place to be good T. melanosporum habitat, and thus of the probability for truffle growers to harvest truffles according to the environmental properties of their truffle orchards.Keywords: dolomite; limestone; mycorrhizae; Quercus ilex; field survey; Tuber melanosporum.

  1. Enhancement of cell viability and alkaline polygalacturonate lyase production by sorbitol co-feeding with methanol in Pichia pastoris fermentation.

    Science.gov (United States)

    Wang, Zhihao; Wang, Yun; Zhang, Dongxu; Li, Jianghua; Hua, Zhaozhe; Du, Guocheng; Chen, Jian

    2010-02-01

    Alkaline polygalacturonate lyase (PGL) production by Pichia pastoris GS115 was used as a model to study the mechanism and strategy for enhancing heterologous protein production. In order to enhance cell viability and volumetric recombinant protein productivity, sorbitol, which had been confirmed to be a non-repressive carbon source, was added together with methanol during the induction phase. The resultant PGL activity was up to 1593 U mL(-1), which was enhanced 1.85-fold compared to the control (863 U mL(-1)) cultured with sorbitol added at a constant rate of 3.6 g h(-1)L(-1) after an induction period of 100 h. Further results revealed that an appropriate sorbitol co-feeding strategy not only decreased the cell mortality to 8.8% (the control is about 23.1%) in the end of fermentation, but also reduced the proteolytic degradation of PGL.

  2. 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.

  3. 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

  4. Temperature profile during the alkaline dissolution of Al for the production of Mo-99

    Energy Technology Data Exchange (ETDEWEB)

    Araujo, Izilda C.; Camilo, Ruth L.; Mindrisz, Ana C.; Forbicini, Christina A.L.G. de O., E-mail: cruzaraujo22@gmail.com, E-mail: rcamilo@ipen.br, 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

    Since September 2008 Brazil is developing the project called Brazilian Multipurpose Reactor (RMB), having as main objective to produce about 1000 Ci/week of {sup 99}Mo. The {sup 99m}Tc, daughter 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. This study is part of the project to obtain {sup 99}Mo by alkaline dissolution of UAl{sub x}-Al targets. Al corresponds to about 79% of the total weight of the UAl{sub x}-Al target. The initial reaction temperature is an important parameter, since it has great influence on the value of the maximum temperature and dissolution time. The heat of reaction must be removed from the dissolving system by controlled cooling. According to literature as a safety condition, the dissolution process must have its temperature controlled so that the maximum temperature to be around 90 deg C.The behavior of the temperature during dissolution using a thermostatic bath with continuous circulation to maintain its value at around 90 deg C was studied. The alkaline solution of NaOH{sub 3} mol.L{sup -1} and NaNO{sub 3} 2 mol.L{sup -1}. As initial temperatures were: 70, 75 and 80 deg C and initial temperatures of the thermostatic bath were: 40,45, 50, 60 and 70 deg C. The results indicate that none of the studies it was possible to maintain the temperature of dissolution near 90 deg C. In the studies where the maximum temperature was around 93 deg C dissolution was incomplete. It may be necessary an intermittent cooling rather than continuous to ensure that the temperature profile is maintained around 90 deg C. (author)

  5. Production of bioactive peptide hydrolysates from deer, sheep and pig plasma using plant and fungal protease preparations.

    Science.gov (United States)

    Bah, Clara S F; Bekhit, Alaa El-Din A; Carne, Alan; McConnell, Michelle A

    2015-06-01

    Plasma separated from deer, sheep and pig blood, obtained from abattoirs, was hydrolysed using protease preparations from plant (papain and bromelain) and fungal (FP400 and FPII) sources. Antioxidant and antimicrobial activities of the peptide hydrolysates obtained after 1, 2, 4 and 24h of hydrolysis, were investigated. The release of trichloroacetic acid-soluble peptides over the hydrolysis period was monitored using the o-phthaldialdehyde (OPA) assay, while the hydrolysis profiles were visualised using SDS-PAGE. The major plasma proteins in the animal plasmas were identified using MALDI-TOF-TOF MS. Hydrolysates of plasma generated with fungal proteases exhibited higher DPPH radical-scavenging, oxygen radical-scavenging capacity (ORAC) and ferric reducing antioxidant power (FRAP) than those generated with plant proteases for all three animal plasmas. No antimicrobial activity was detected in the hydrolysates. The results indicated that proteolytic hydrolysis of animal blood plasmas, using fungal protease preparations in particular, produces hydrolysates with high antioxidant properties.

  6. Principles of technological design of wasteless chemical processes based on the use of wastes for production of alkaline slag cements and concretes

    Energy Technology Data Exchange (ETDEWEB)

    Glukhovskii, V.D.; Chernobaev, I.P.; Emel' yanov, B.M.; Semenyuk, A.P.

    1985-05-20

    The strength characteristics of alkaline slag-cement made with the use of waste from alkaline sealing of metals are presented. The cement was prepared from granulated blast-furnance slag with average component contents in the following ranges (mass %): SiO/sub 2/ 36.0-40.2, Al/sub 2/O/sub 3/ 4-18.2, FeO 0.1-3.7, MnO 0.4-5.2, CaO 33.1-48.8, MgO 2.2-9.8. With the use of wastes from the descaling process in alkali melts for production of alkaline slag cements it is possible to obtain highly effective cements of type 700-900, which is 2 to 3 times the value for portland cements. Therefore, the use of wastes from alkaline descaling for production of alkaline slag cements is of great economic and conservational significance. It is possible to devise a wasteless process of scale removal from metals; this is an important advantage of the alkaline scaling method over acid pickling.

  7. 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.

  8. In-situ Alkaline Transesterification of Jatropha curcas seed Oil for Production of Biodiesel and Nontoxic Jatropha seed Cake

    Directory of Open Access Journals (Sweden)

    Novizar Nazir

    2014-01-01

    Full Text Available 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 concentration in methanol, 171:1 methanol/oil mole ratio, 45.66 oC reaction temperature and 3.02 h reaction time. The use of alkaline methanol as extraction and reaction solvent, which would be useful for extraction oil and phorbol esters, would reduce the phorbol esters content in the Jatropha curcas seed cake. The cake after in-situ transesterification is rich in protein and is a potential source of livestock feed. Further, the the toxicity studies were also investigated on male rate by feeding the seed cake after after in-situ transesterification as well as the from solvent and mechanical extraction. Food intake, growth rate, protein efficiency ratio (PER and transformation index (TI showed that the meal is potential as protein supplement to livestock feed.

  9. 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.

  10. A statistical approach for optimization of alkaline lipase production by ascidian associated—Halobacillus trueperi RSK CAS9

    Directory of Open Access Journals (Sweden)

    Ramamoorthy Sathishkumar

    2015-12-01

    Full Text Available A marine ascidian-associated bacterium, Halobacillus trueperi RSK CAS9, was optimized for lipase production by response surface methodology using marine waste as substrate. The central composite design was employed, and the optimal medium constituents for maximum lipase production (1355.81 U/ml were determined to be tuna powder (14.58 g/l, olive oil (5.05 ml/l; NaCl (72.42 g/l, temperature (45 °C and pH 9.0. An alkaline lipase was purified to 8.46 fold with 1193.59 U mg−1 specific activities with the molecular weight of 44 kDa. The activity was substantially inhibited by EDTA and PMSF, indicating that it was a metalloenzyme serine residue which was essential for catalytic activity. Thus, lipase production by microbial conversion of marine fish wastes in this study suggested its potential utilization for the production of high value products.

  11. Characterisation of the Thermostable Protease AprX in Strains of Pseudomonas Fluorescens and Impact on the Shelf-life of Dairy Products: Preliminary Results.

    Science.gov (United States)

    Andreani, Nadia Andrea; Carraro, Lisa; Fasolato, Luca; Balzan, Stefania; Lucchini, Rosaria; Novelli, Enrico; Cardazzo, Barbara

    2016-09-20

    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.

  12. 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.

  13. "Supergreen" Renewables: Integration of Mineral Weathering Into Renewable Energy Production for Air CO2 Removal and Storage as Ocean Alkalinity

    Science.gov (United States)

    Rau, G. H.; Carroll, S.; Ren, Z. J.

    2015-12-01

    Excess planetary CO2 and accompanying ocean acidification are naturally mitigated on geologic time scales via mineral weathering. Here, CO2 acidifies the hydrosphere, which then slowly reacts with silicate and carbonate minerals to produce dissolved bicarbonates that are ultimately delivered to the ocean. This alkalinity not only provides long-term sequestration of the excess atmospheric carbon, but it also chemically counters the effects of ocean acidification by stabilizing or raising pH and carbonate saturation state, thus helping rebalance ocean chemistry and preserving marine ecosystems. Recent research has demonstrated ways of greatly accelerating this process by its integration into energy systems. Specifically, it has been shown (1) that some 80% of the CO2 in a waste gas stream can be spontaneously converted to stable, seawater mineral bicarbonate in the presence of a common carbonate mineral - limestone. This can allow removal of CO2 from biomass combustion and bio-energy production while generating beneficial ocean alkalinity, providing a potentially cheaper and more environmentally friendly negative-CO2-emissions alternative to BECCS. It has also been demonstrated that strong acids anodically produced in a standard saline water electrolysis cell in the formation of H2 can be reacted with carbonate or silicate minerals to generate strong base solutions. These solutions are highly absorptive of air CO2, converting it to mineral bicarbonate in solution. When such electrochemical cells are powered by non-fossil energy (e.g. electricity from wind, solar, tidal, biomass, geothermal, etc. energy sources), the system generates H2 that is strongly CO2-emissions-negative, while producing beneficial marine alkalinity (2-4). The preceding systems therefore point the way toward renewable energy production that, when tightly coupled to geochemical mitigation of CO2 and formation of natural ocean "antacids", forms a high capacity, negative-CO2-emissions, "supergreen

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

    Energy Technology Data Exchange (ETDEWEB)

    Mindrisz, Ana C.; Camilo, Ruth L.; Araujo, Izilda C.; Forbicini, Christina A.L.G. de O., E-mail: acmindri@ipen.br, E-mail: rcamilo@ipen.br, E-mail: cruzaraujo22@gmail.com, E-mail: cforbici@ipen.br [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil)

    2013-07-01

    Since 2008, due to the global crisis in the production of radioisotope {sup 99}Mo, which product of decay, {sup 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{sup 9M}o from irradiated UAl{sub x}-Al LEU targets are under development. Processing time should be minimized, considering the short half-life of {sup 99}Mo and {sup 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{sub x}-Al targets. Dissolution time and gas releasing were evaluated using the following alkaline solutions: a) NaOH 3 mol.L{sup -1} and NaNO{sub 3} 2 mol.L{sup -1}, b) NaOH 3 mol.L{sup -1} and NaNO{sub 3} 4 mol.L{sup -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{sub 3} 2 mol.L{sup -1} and 104.55% for NaNO{sub 3} 4 mol.L{sup -1}. The dissolution with NaNO{sub 3} 2 mol.L{sup -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{sub 3} 4 mol.L{sup -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)

  15. Producción Fúngica de Proteasas Inducidas con Pelo de Cerdo Fungal Production of Proteases Induced with Pig Hair

    Directory of Open Access Journals (Sweden)

    Imir R Vázquez

    2008-01-01

    Full Text Available Se evaluaron las condiciones de producción de proteasas fúngicas inducidas con pelo de cerdo usando dos sistemas de cultivo, el proceso de cultivo en estado sólido y el cultivo líquido sumergido. El medio de cultivo fue Czapek-Dox en ambos casos. La cinética fue monitoreada cada 24 horas durante un periodo de incubación de 120 horas. La actividad proteolítica fue evaluada por hidrólisis de azocaseína y la producción de la biomasa fue estimada por el método gravimétrico. Rhizopus oryzae fue el microorganismo capaz de utilizar el pelo de cerdo como única fuente de carbono-nitrógeno, produciendo en cultivo sumergido los mayores títulos de actividad queratinasa. Se determinó el efecto que tiene el pH, el pretratamiento del sustrato y la adición de diferentes niveles de material queratinoso sobre el crecimiento fúngico y la actividad enzimática. Los resultados permitieron obtener títulos de actividad proteolítica de 975 U/L.The production of new proteases induced with pig hair was evaluated using two systems, submerged culture and solid state culture. Culture medium was Czapek-Dox for both systems. Enzyme production kinetic was monitored each 24 hours during 120 hours. Protelytic activity was evaluated with the azocaseín method and biomass production was determined by the gravimetric method. Rhizopus oryzae was the microorganism with the capacity of using the pig hair as the single carbon-nitrogen source, producing in submerged fermentation the highest enzymatic titers. The effect of pH, the substrate pretreatment and addition of different levels of substrate on proteolyitic activity and biomass production were determined. Results permitted to obtain titers of proteolytic activity of 975 U/L.

  16. Proteases in Periodontal Disease

    Directory of Open Access Journals (Sweden)

    Ana Rita Sokolonski ANTON

    2006-09-01

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

  17. Alkaline inulinase production by a newly isolated bacterium Marinimicrobium sp. LS-A18 and inulin hydrolysis by the enzyme.

    Science.gov (United States)

    Li, Ai-Xia; Guo, Li-Zhong; Lu, Wei-Dong

    2012-01-01

    To date, all of microbial inulinases reported showed optimal activity at pH values ranging from 3.5 to 7.0. A bacterial strain, Marinimicrobium sp. LS-A18, showing high extracellular inulinolytic activity was isolated from a marine solar saltern of the Yellow Sea in China. Maximum enzyme activity was obtained at 55°C and pH 9.0, respectively. The inulinase activity was induced by inulin, but not by the other carbon sources employed. Under the optimal medium and culture condition, the highest inulinase activity, 14.6 U/ml, was obtained after 96 h of incubation at shake flask level. The optimal medium for inulinase production was MHI medium containing 4% inulin, 1% peptone and 5% NaCl, while the optimal culture condition for inulinase production were pH 7.5, temperature 37°C, agitation speed 210 rpm, medium volume 40 ml in 250 ml shake flask, and incubation time 96 h. A large amount of monosaccharides was released after inulin hydrolysis by the inulinase from strain LS-A18. This is the first report on alkaline inulinase production from microorganism.

  18. Augmentation of protein-derived acetic acid production by heat-alkaline-induced changes in protein structure and conformation.

    Science.gov (United States)

    Wang, Xu; Li, Yanbo; Liu, Junxin; Ren, Nan-Qi; Qu, Jiuhui

    2016-01-01

    Waste-derived acetic acid (HAc) is an attractive feedstock for microbe-mediated biofuel production. However, fermentative conversion of HAc from waste-activated sludge (WAS) has low yield because of the high concentration of proteins not readily utilizable by microorganisms without prior hydrolysis. We investigated a combined technology for HAc augmentation during sludge protein fermentation. The maximal HAc yield increased over two-fold, reaching 0.502 ± 0.021 g/g protein (0.36 ± 0.01 g COD/g COD, ∼52% of the total volatile fatty acids) when synthetic sludge protein was heated at 120 °C for 30 min, treated at pH 12 for 24 h, and fermented at pH 9 for 72 h. Comprehensive analysis illustrated that the heat-alkaline pretreatment significantly induced protein fragmentation, simultaneously increasing the efficiency of protein biohydrolysis (from 35.5% to 85.9%) by inducing conformational changes indicative of protein unfolding. Consequently, the native α-helix content was decreased from 67.3% to 32.5% by conversion to an unordered shape, whose content increased from 27.5% to 45.5%; disulfide bonds were cleaved, whereas the main S-S stretching pattern was altered from gauche-gauche-gauche to gauche-gauche-trans, consequently causing increased protein susceptibility to proteolytic hydrolysis (76.3% vs. 47.0%). Economic analysis indicated that anaerobic fermentation with appropriate heat-alkaline pretreatment is a cost-effective approach for waste conversion to energy sources such as HAc.

  19. The "Bridge" in the Epstein-Barr virus alkaline exonuclease protein BGLF5 contributes to shutoff activity during productive infection.

    Science.gov (United States)

    Horst, Daniëlle; Burmeister, Wim P; Boer, Ingrid G J; van Leeuwen, Daphne; Buisson, Marlyse; Gorbalenya, Alexander E; Wiertz, Emmanuel J H J; Ressing, Maaike E

    2012-09-01

    Replication of the human herpesvirus Epstein-Barr virus drastically impairs cellular protein synthesis. This shutoff phenotype results from mRNA degradation upon expression of the early lytic-phase protein BGLF5. Interestingly, BGLF5 is the viral DNase, or alkaline exonuclease, homologues of which are present throughout the herpesvirus family. During productive infection, this DNase is essential for processing and packaging of the viral genome. In contrast to this widely conserved DNase activity, shutoff is only mediated by the alkaline exonucleases of the subfamily of gammaherpesviruses. Here, we show that BGLF5 can degrade mRNAs of both cellular and viral origin, irrespective of polyadenylation. Furthermore, shutoff by BGLF5 induces nuclear relocalization of the cytosolic poly(A) binding protein. Guided by the recently resolved BGLF5 structure, mutants were generated and analyzed for functional consequences on DNase and shutoff activities. On the one hand, a point mutation destroying DNase activity also blocks RNase function, implying that both activities share a catalytic site. On the other hand, other mutations are more selective, having a more pronounced effect on either DNA degradation or shutoff. The latter results are indicative of an oligonucleotide-binding site that is partially shared by DNA and RNA. For this, the flexible "bridge" that crosses the active-site canyon of BGLF5 appears to contribute to the interaction with RNA substrates. These findings extend our understanding of the molecular basis for the shutoff function of BGLF5 that is conserved in gammaherpesviruses but not in alpha- and betaherpesviruses.

  20. Biotechnology of cold-active proteases.

    Science.gov (United States)

    Joshi, Swati; Satyanarayana, Tulasi

    2013-05-03

    The bulk of Earth's biosphere is cold (<5 °C) and inhabited by psychrophiles. Biocatalysts from psychrophilic organisms (psychrozymes) have attracted attention because of their application in the ongoing efforts to decrease energy consumption. Proteinases as a class represent the largest category of industrial enzymes. There has been an emphasis on employing cold-active proteases in detergents because this allows laundry operations at ambient temperatures. Proteases have been used in environmental bioremediation, food industry and molecular biology. In view of the present limited understanding and availability of cold-active proteases with diverse characteristics, it is essential to explore Earth's surface more in search of an ideal cold-active protease. The understanding of molecular and mechanistic details of these proteases will open up new avenues to tailor proteases with the desired properties. A detailed account of the developments in the production and applications of cold-active proteases is presented in this review.

  1. Biotechnology of Cold-Active Proteases

    Directory of Open Access Journals (Sweden)

    Tulasi Satyanarayana

    2013-05-01

    Full Text Available The bulk of Earth’s biosphere is cold (<5 °C and inhabited by psychrophiles. Biocatalysts from psychrophilic organisms (psychrozymes have attracted attention because of their application in the ongoing efforts to decrease energy consumption. Proteinases as a class represent the largest category of industrial enzymes. There has been an emphasis on employing cold-active proteases in detergents because this allows laundry operations at ambient temperatures. Proteases have been used in environmental bioremediation, food industry and molecular biology. In view of the present limited understanding and availability of cold-active proteases with diverse characteristics, it is essential to explore Earth’s surface more in search of an ideal cold-active protease. The understanding of molecular and mechanistic details of these proteases will open up new avenues to tailor proteases with the desired properties. A detailed account of the developments in the production and applications of cold-active proteases is presented in this review.

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

    Institute of Scientific and Technical Information of China (English)

    王国泽; 袁怀波; 徐建文

    2012-01-01

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

  3. Impact of aeration and agitation on metabolic heat and protease secretion of Aspergillus tamarii in a real-time biological reaction calorimeter.

    Science.gov (United States)

    Dhandapani, Balaji; Mahadevan, Surianarayanan; Dhilipkumar, Sathish Sundar; Rajkumar, Suseela; Mandal, Asit Baran

    2012-06-01

    The effects of aeration and agitation on metabolic heat, alkaline protease production and morphology for Aspergillus tamarii MTCC5152 are reported in this manuscript. Measurement of metabolic heat has been attempted by the continuous and dynamic heat balance method in a biological real-time reaction calorimeter. At lower agitation intensities, growth-related processes were dominating. As a result the protease activity and the product heat yields were lower than those for 350 and 450 rpm. Although biomass growth was necessary to obtain maximum protease yield, agitation seemed to play a vital role in the protease production process. Energy dissipation per circulation function of the process is also deduced from power input. At optimal conditions, 350 rpm and 1 vvm, the gassed power required was 0.133 W. Pellet morphology and protease production were studied under different aeration and agitation intensities of A. tamarii. Pellet structure was considerably influenced by DO, a higher DO level resulted in denser pellets (1,018.4 kg/m(3)) leading to higher protease activity. Coupling of hydrodynamics and bio-reaction highlighted the complex relationship between energy dissipation, substrate uptake rate and fungal physiology. This study emphasised the potential of biocalorimetry as a reliable monitoring and robust control tool for aerobic fermentation of A. tamarii, using agricultural by-products.

  4. 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.

  5. Analysis of by-product formation and sugar monomerization in sugarcane bagasse pretreated at pilot plant scale: Differences between autohydrolysis, alkaline and acid pretreatment

    NARCIS (Netherlands)

    Pol, van der E.C.; Bakker, R.; Zeeland, van A.N.T.; Sanchez Garcia, D.; Punt, A.M.; Eggink, G.

    2015-01-01

    Sugarcane bagasse is an interesting feedstock for the biobased economy since a large fraction is polymerized sugars. Autohydrolysis, alkaline and acid pretreatment conditions combined with enzyme hydrolysis were used on lignocellulose rich bagasse to acquire monomeric. By-products found after pretre

  6. Optimization of a multi-stage, multi-subunit malaria vaccine candidate for the production in Pichia pastoris by the identification and removal of protease cleavage sites.

    Science.gov (United States)

    Spiegel, Holger; Schinkel, Helga; Kastilan, Robin; Dahm, Pia; Boes, Alexander; Scheuermayer, Matthias; Chudobová, Ivana; Maskus, Dominika; Fendel, Rolf; Schillberg, Stefan; Reimann, Andreas; Fischer, Rainer

    2015-04-01

    We demonstrated the successful optimization of a recombinant multi-subunit malaria vaccine candidate protein for production in the methylotrophic yeast Pichia pastoris by the identification and subsequent removal of two protease cleavage sites. After observing protein degradation in the culture supernatant of a fed-batch fermentation, the predominant proteolytic fragment of the secreted recombinant protein was analyzed by mass spectrometry. The MS data indicated the cleavage of an amino acid sequence matching the yeast KEX2-protease consensus motif EKRE. The cleavage in this region was completely abolished by the deletion of the EKRE motif in a modified variant. This modified variant was produced, purified, and used for immunization of rabbits, inducing high antigen specific antibody titers (2 × 10(6) ). Total IgG from rabbit immune sera recognized different stages of Plasmodium falciparum parasites in immunofluorescence assays, indicating native folding of the vaccine candidate. However, the modified variant was still degraded, albeit into different fragments. Further analysis by mass spectrometry and N-terminal sequencing revealed a second cleavage site downstream of the motif PEVK. We therefore removed a 17-amino-acid stretch including the PEVK motif, resulting in the subsequent production of the full-length recombinant vaccine candidate protein without significant degradation, with a yield of 53 mg per liter culture volume. We clearly demonstrate that the proteolytic degradation of recombinant proteins by endogenous P. pastoris proteases can be prevented by the identification and removal of such cleavage sites. This strategy is particularly relevant for the production of recombinant subunit vaccines, where product yield and stability play a more important role than for the production of a stringently-defined native sequence which is necessary for most therapeutic molecules.

  7. 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

    (corn cobs) which compared favorably to those reported for the other microorganisms. Use of de-esterified corn cobs as carbon source decreased FAE production by 5.5-fold compared to untreated corn cobs even though ferulic acid (FA) was added to the concentration found in alkali-extracts of corn cobs......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...

  8. Extracellular proteases of Halobacillus blutaparonensis strain M9, a new moderately halophilic bacterium

    Directory of Open Access Journals (Sweden)

    Anderson F. Santos

    2013-12-01

    Full Text Available Halophilic microorganisms are source of potential hydrolytic enzymes to be used in industrial and/or biotechnological processes. In the present study, we have investigated the ability of the moderately halophilic bacterium Halobacillus blutaparonensis (strain M9, a novel species described by our group, to release proteolytic enzymes. This bacterial strain abundantly proliferated in Luria-Bertani broth supplemented with 2.5% NaCl as well as secreted proteases to the extracellular environment. The production of proteases occurred in bacterial cells grown under different concentration of salt, ranging from 0.5% to 10% NaCl, in a similar way. The proteases secreted by H. blutaparonensis presented the following properties: (i molecular masses ranging from 30 to 80 kDa, (ii better hydrolytic activities under neutral-alkaline pH range, (iii expression modulated according to the culture age, (iv susceptibility to phenylmethylsulphonyl fluoride, classifying them as serine-type proteases, (v specific cleavage over the chymotrypsin substrate, and (vi enzymatic stability in the presence of salt (up to 20% NaCl and organic solvents (e.g., ether, isooctane and cyclohexane. The proteases described herein are promising for industrial practices due to its haloalkaline properties.

  9. Extracellular proteases of Halobacillus blutaparonensis strain M9, a new moderately halophilic bacterium

    Science.gov (United States)

    Santos, Anderson F.; Valle, Roberta S.; Pacheco, Clarissa A.; Alvarez, Vanessa M.; Seldin, Lucy; Santos, André L.S.

    2013-01-01

    Halophilic microorganisms are source of potential hydrolytic enzymes to be used in industrial and/or biotechnological processes. In the present study, we have investigated the ability of the moderately halophilic bacterium Halobacillus blutaparonensis (strain M9), a novel species described by our group, to release proteolytic enzymes. This bacterial strain abundantly proliferated in Luria-Bertani broth supplemented with 2.5% NaCl as well as secreted proteases to the extracellular environment. The production of proteases occurred in bacterial cells grown under different concentration of salt, ranging from 0.5% to 10% NaCl, in a similar way. The proteases secreted by H. blutaparonensis presented the following properties: (i) molecular masses ranging from 30 to 80 kDa, (ii) better hydrolytic activities under neutral-alkaline pH range, (iii) expression modulated according to the culture age, (iv) susceptibility to phenylmethylsulphonyl fluoride, classifying them as serine-type proteases, (v) specific cleavage over the chymotrypsin substrate, and (vi) enzymatic stability in the presence of salt (up to 20% NaCl) and organic solvents (e.g., ether, isooctane and cyclohexane). The proteases described herein are promising for industrial practices due to its haloalkaline properties. PMID:24688526

  10. [Analysis of alkaline CuO degradation products of acid detergent fiber from tobacco leaves by using liquid chromatography].

    Science.gov (United States)

    Hao, Weiqiang; Wang, Leijun; Wu, Shun; Yue, Bangyi; Chen, Qiang; Zhang, Peipei

    2015-07-01

    The acid detergent fiber (ADF) from tobacco leaves was obtained by treating the sample with petroleum ether-ethanol (6:4, v/v), 30 g/L sodium dodecylsulfate and 0.5 mol/L sulphuric acid containing 20 g/L hexadecyl trimethyl ammonium bromide successively. The ADF was degraded by the alkaline CuO oxidation procedure. In this work, six samples of ADF degradation products from tobacco leaves were prepared. The samples were analyzed by using gradient liquid chromatography (LC) where an Ultimate XB C18 column was used as stationary phase, with a mixture of methanol and water as mobile phase, at a column temperature of 35 °C and a flow rate of 0.8 mL/min. Dual wavelengths of 280 nm and 320 nm were chosen for the detection. It was found that there were four characteristic peaks for the ADF degradation products. By taking these peaks as research object, the optimum time for the degradation was found to be 5 h and the sample solution could be kept stable within 7 days. The established method may provide a new approach for the studies of the differences between lignin composition in different tobacco leaves and the relationship between lignin content and the smoking quality of tobacco leaves.

  11. 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)

  12. 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.

  13. Lectrochemical promotion of novel catalysts with alkaline conductors for hydrogen production from methanol

    OpenAIRE

    González Cobos, Jesús

    2016-01-01

    Hydrogen is a very important feedstock in the chemical industry and a promising energy carrier with main application in internal combustion engines and fuel cell technology as an alternative to the massive consumption of fossil fuels. H2 presents a high gravimetric energy density and can be considered as a clean synthetic fuel depending on the sustainability of the energy and raw material employed for its production. Hydrogen is currently obtained mainly via methane steam reforming. However, ...

  14. 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)

  15. The Spl Serine Proteases Modulate Staphylococcus aureus Protein Production and Virulence in a Rabbit Model of Pneumonia

    Science.gov (United States)

    Salgado-Pabon, Wilmara; Meyerholz, David K.; White, Mark J.; Schlievert, Patrick M.

    2016-01-01

    ABSTRACT The Spl proteases are a group of six serine proteases that are encoded on the νSaβ pathogenicity island and are unique to Staphylococcus aureus. Despite their interesting biochemistry, their biological substrates and functions in virulence have been difficult to elucidate. We found that an spl operon mutant of the community-associated methicillin-resistant S. aureus USA300 strain LAC induced localized lung damage in a rabbit model of pneumonia, characterized by bronchopneumonia observed histologically. Disease in the mutant-infected rabbits was restricted in distribution compared to that in wild-type USA300-infected rabbits. We also found that SplA is able to cleave the mucin 16 glycoprotein from the surface of the CalU-3 lung cell line, suggesting a possible mechanism for wild-type USA300 spreading pneumonia to both lungs. Investigation of the secreted and surface proteomes of wild-type USA300 and the spl mutant revealed multiple alterations in metabolic proteins and virulence factors. This study demonstrates that the Spls modulate S. aureus physiology and virulence, identifies a human target of SplA, and suggests potential S. aureus targets of the Spl proteases. IMPORTANCE Staphylococcus aureus is a versatile human pathogen that produces an array of virulence factors, including several proteases. Of these, six proteases called the Spls are the least characterized. Previous evidence suggests that the Spls are expressed during human infection; however, their function is unknown. Our study shows that the Spls are required for S. aureus to cause disseminated lung damage during pneumonia. Further, we present the first example of a human protein cut by an Spl protease. Although the Spls were predicted not to cut staphylococcal proteins, we also show that an spl mutant has altered abundance of both secreted and surface-associated proteins. This work provides novel insight into the function of Spls during infection and their potential ability to degrade

  16. A novel alkaline lipase from Ralstonia with potential application in biodiesel production.

    Science.gov (United States)

    Yoo, Hah-Young; Simkhada, Jaya Ram; Cho, Seung Sik; Park, Don Hee; Kim, Seung Wook; Seong, Chi Nam; Yoo, Jin Cheol

    2011-05-01

    With the aim of isolating a biocatalyst able to catalyze biodiesel production from microbial source, Ralstonia sp. CS274 was isolated and a lipase from the strain (RL74) was purified. Molecular weight of RL74 was estimated to be 28,000 Da by SDS-PAGE. The activity was highest at 50-55°C and pH 8.0-9.5 and was stable at pH 7.0-12.0 and up to 45°C. It was resistant to oxidizing and reducing agents and the activity was enhanced by detergents. RL74 was 1,3 specific and K(m) and V(max) for p-nitrophenyl palmitate were 2.73 ± 0.6mM and 101.4 ± 1.9 mM/min mg, respectively. N-terminal amino acid sequence showed partial homology with that of Penicillium lipases. RL74 produced biodiesel more efficiently in palm oil than in soybean oil; and the production was highest at pH 8.0, at 5% methanol and at 20% water content.

  17. Prevention of ARD through stabilization of waste rock with alkaline by-products : results from a meso-scale experiment

    Energy Technology Data Exchange (ETDEWEB)

    Backstrom, M.; Allard, B. [Orebro Univ. (Sweden). Man-Technology Environment Research Centre; Sartz, L.; Karlsson, S. [Orebro Univ. (Sweden). Man-Technology Environment Research Centre; Bergskraft Bergslagen, Kopparberg (Sweden)

    2010-07-01

    Mine waste can be mixed with alkaline materials to neutralize and increase the immobilization of trace elements. An impermeable layer can also be created if the alkaline additions react with the waste to form hardpans. Alkaline injection processes have been used in the western United States, where approximately 2 to 3.5 million tonnes of mine tailings have been limed with calcite, calcium hydroxide (Ca(OH){sub 2}), and calcium oxide (CaO). In this study, stabilization experiments were conducted to simulate conditions where weathered mine waste was mixed with various alkaline materials. The aim of the study was to determine the optimal conditions for preventing the oxidation of mine waste isolating surfaces. The alkaline materials included fly ash, lime mud, green liquor dregs, and lime kiln dust. The mine waste and alkaline materials were layered in barrels. Expanded clay aggregates were used to minimize the risk of clogging. Results of the experiments showed that the pH in the alkaline-treated systems increased between 1.3 and 27 pH units when compared with untreated reference samples. The increased pH resulted in a decrease in trace element concentrations of approximately 96 percent. The samples containing fly ash performed better than other systems. 4 refs., 1 tab., 2 figs.

  18. 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.

  19. Production of Bioactive Peptides from Soybean Meal by Solid State Fermentation with Lactic Acid Bacteria and Protease

    Directory of Open Access Journals (Sweden)

    Naifu Wang

    2014-10-01

    Full Text Available In this study, soybean meal was first solid state fermented with different strains of Lactic Acid Bacteria (LAB. Among the strains used, Lactobacillus plantarum Lp6 was selected for further studies because of its highest Degree of Hydrolysis (DH of protein (2.49±0.08% in soybean meal after 72 h fermentation. Soybean meal fermented with L. plantarum Lp6 can also improve its DPPH radical scavenging and Angiotensin Converting Enzyme (ACE inhibitory activities. The addition of protease into soybean meal during the fermentation resulted in lowered IC50 of DPPH radical scavenging and ACE inhibitory activities, indicating more bioactive peptides were produced during fermentation. Molecular weight distribution analysis revealed the Extracts from Fermented Soybean Meal (EFSM was mainly composed of oligopeptides. These results indicated that soybean meal fermented with L. plantarum Lp6 and protease could be an easy and cheap method to produce functional food.

  20. Profiling acylated homoserine lactones in Yersinia ruckeri and influence of exogenous acyl homoserine lactones and known quorum-sensing inhibitors on protease production

    DEFF Research Database (Denmark)

    Kastbjerg, Vicky Gaedt; Nielsen, Kristian Fog; Dalsgaard, Inger;

    2007-01-01

    produced at least eight different acylated homoserine lactones (AHLs) with N-(3-oxooctanoyl)-l-homoserine lactone (3-oxo-C8-HSL) being the dominant molecule. Also, some uncommon AHL, N-(3-oxoheptanoyl)-l-homoserine lactone (3-oxo-C7-HSL) and N-(3-oxononanoyl)-l-homoserine lactone (3-oxo-C9-HSL), were...... produced. 3-oxo-C8-HSL was detected in organs from fish infected with Y. ruckeri. Protease production was significantly lower at temperatures above 23 degrees C than below although growth was faster at the higher temperatures. Neither addition of sterile filtered high-density Y. ruckeri culture supernatant...

  1. Low dietary copper increases fecal free radical production, fecal water alkaline phosphatase activity and cytotoxicity in healthy men.

    Science.gov (United States)

    Davis, Cindy D

    2003-02-01

    One possible dietary factor that may increase susceptibility to colon cancer is inadequate copper intake. The objective of this study was to investigate the effects of low and adequate copper intakes on copper nutriture and putative risk factors for colon cancer susceptibility in healthy men. Seventeen healthy free-living nonsmoking men aged 21-52 y completed a 13-wk controlled feeding study in a randomized crossover design. The basal diet contained 0.59 mg Cu/13.65 MJ. After a 1-wk equilibration period in which the men consumed the basal diet supplemented with 1.0 mg Cu/d, they were randomly assigned to receive either the basal diet or the basal diet supplemented with 2 mg Cu/d for 6 wk. After the first dietary period, the men immediately began to consume the other level of Cu for the last 6 wk. They collected their feces during the equilibration period and during the last 2 wk of the two dietary periods for free radical and fecal water analysis. Low dietary copper significantly (P copper significantly (P copper concentrations but did not affect fecal water volume, pH, iron or zinc concentrations. In contrast to the fecal analysis, hematological indicators of copper status were not significantly affected by the dietary treatments. These results suggest that low dietary copper adversely affects fecal free radical production and fecal water alkaline phosphatase activity, which are putative risk factors for colon cancer.

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

    Directory of Open Access Journals (Sweden)

    Salcedo L.

    1998-06-01

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

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

    Science.gov (United States)

    Šustr, Vladimír; Stingl, Ulrich; Brune, Andreas

    2014-08-01

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

  4. Alkaline Pretreatment of Sugarcane Bagasse and Filter Mud Codigested to Improve Biomethane Production

    Science.gov (United States)

    Mehryar, Esmaeil; Bi, Jinhua

    2016-01-01

    To enhance the codigestion of degradation and improve biomethane production potential, sugarcane bagasse and filter mud were pretreated by sodium hydroxide NaOH 1 N at 100°C for 15, 30, and 45 minutes, respectively. Biomethane generation from 1-liter batch reactor was studied at mesophilic temperature (37 ± 1)°C, solid concentrations of 6%, and five levels of mixing proportion with and without pretreatment. The results demonstrate that codigestion of filter mud with bagasse produces more biomethane than fermentation of filter mud as single substrate; even codigested substrate composition presented a better balance of nutrients (C/N ratio of 24.70) when codigestion ratio between filter mud and bagasse was 25 : 75 in comparison to filter mud as single substrate (C/N ratio 9.68). All the pretreatments tested led to solubilization of the organic matter, with a maximum lignin reduction of 86.27% and cumulative yield of biomethane (195.8 mL·gVS−1, digestion of pretreated bagasse as single substrate) obtained after 45 minutes of cooking by NaOH 1 N at 100°C. Under this pretreatment condition, significant increase in cumulative methane yield was observed (126.2 mL·gVS−1) at codigestion ratio of 25 : 75 between filter mud and bagasse by increase of 81.20% from untreated composition.

  5. Alkaline pretreatment for enhancement of biogas production from banana stem and swine manure by anaerobic codigestion.

    Science.gov (United States)

    Zhang, Chengming; Li, Jihong; Liu, Chen; Liu, Xiaoling; Wang, Jianlong; Li, Shizhong; Fan, Guifang; Zhang, Lei

    2013-12-01

    The objective of this research was to propose and investigate the availability of digested banana stem (BS) to produce biogas. Squeezed BS with less moisture content was used for biogas production through a combination of NaOH pretreatment, solid-state fermentation, and codigestion technologies. NaOH doses were optimized according to biogas fermentation performance, and the best dose was 6% (by weight) based on the total solid (TS) of BS. Under this condition, the lignin, cellulose, and hemicellulose contents decreased from 18.36%, 32.36% and 14.6% to 17.10%, 30.07%, and 10.65%, respectively, after pretreatment. After biogas digestion, TS and volatile solid (VS) reductions of the codigestion were 48.5% and 70.4%, respectively, and the biogas and methane yields based on VS loading were 357.9 and 232.4 mL/g, which were 12.1% and 21.4%, respectively, higher than the control. Results indicated that the proposed process could be an effective method for using BS to produce biogas.

  6. 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.

  7. Protease gene families in Populus and Arabidopsis

    Directory of Open Access Journals (Sweden)

    Jansson Stefan

    2006-12-01

    Full Text Available Abstract Background Proteases play key roles in plants, maintaining strict protein quality control and degrading specific sets of proteins in response to diverse environmental and developmental stimuli. Similarities and differences between the proteases expressed in different species may give valuable insights into their physiological roles and evolution. Results We have performed a comparative analysis of protease genes in the two sequenced dicot genomes, Arabidopsis thaliana and Populus trichocarpa by using genes coding for proteases in the MEROPS database 1 for Arabidopsis to identify homologous sequences in Populus. A multigene-based phylogenetic analysis was performed. Most protease families were found to be larger in Populus than in Arabidopsis, reflecting recent genome duplication. Detailed studies on e.g. the DegP, Clp, FtsH, Lon, rhomboid and papain-Like protease families showed the pattern of gene family expansion and gene loss was complex. We finally show that different Populus tissues express unique suites of protease genes and that the mRNA levels of different classes of proteases change along a developmental gradient. Conclusion Recent gene family expansion and contractions have made the Arabidopsis and Populus complements of proteases different and this, together with expression patterns, gives indications about the roles of the individual gene products or groups of proteases.

  8. Proteases as Insecticidal Agents

    OpenAIRE

    Robert L. Harrison; Bonning, Bryony C.

    2010-01-01

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

  9. Hydrolysis of Fish Protein by Analkaline Protease

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

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

  10. 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.

  11. Assessment of the Fusion Tags on Increasing Soluble Production of the Active TEV Protease Variant and Other Target Proteins in E. coli.

    Science.gov (United States)

    Yu, Xuelian; Sun, Jiaqi; Wang, Weiyu; Jiang, Li; Cheng, Beijiu; Fan, Jun

    2016-12-17

    In this study, five fusion tags affecting soluble production and cleavage activity of the tobacco etch virus (TEV) protease (TEVp) variant in Escherichia coli strains BL21 (DE3) and Rosetta™ (DE3) are investigated. Combination of the augmenting rare transfer RNAs (tRNAs) and the fused expressivity tag (N-terminal seven amino acid residues of E. coli translation initiation factor II) promotes the soluble TEVp partner expressed at relatively high level. Attachment of the maltose-binding protein (MBP) tag increases soluble expression of the protease released from the fusion protein in E. coli cells, but the incorporated TEVp recognition sequence slightly decreases expressivity of the fusion construct. Except for the green fluorescent protein, the attached expressivity tag shows less efficiency than the MBP tag in enhancing expression levels of the selected five target proteins in the Rosetta™ (DE3) cells under different induction conditions. Our results identified that high-level production of the functional target protein as the fusion partner in E. coli is combined with the intrinsic property of fusion tag, fusion protein stability, inherent folding of target protein, rare tRNA abundance, and the incorporated linker. Purified TEVp fusion constructs with the N-terminal expressivity tag, as well as the MBP partner, are the ideal alternatives for removing fusion tag.

  12. Putrescine production via the agmatine deiminase pathway increases the growth of Lactococcus lactis and causes the alkalinization of the culture medium.

    Science.gov (United States)

    del Rio, Beatriz; Linares, Daniel M; Ladero, Victor; Redruello, Begoña; Fernández, Maria; Martin, Maria Cruz; Alvarez, Miguel A

    2015-01-01

    Lactococcus lactis is the most important starter culture organism used in the dairy industry. Although L. lactis species have been awarded Qualified Presumption of Safety status by the European Food Safety Authority, and Generally Regarded as Safe status by the US Food and Drug Administration, some strains can produce the biogenic amine putrescine. One such strain is L. lactis subsp. cremoris CECT 8666 (formerly L. lactis subsp. cremoris GE2-14), which was isolated from Genestoso cheese. This strain catabolizes agmatine to putrescine via the agmatine deiminase (AGDI) pathway, which involves the production of ATP and two ammonium ions. The present work shows that the availability of agmatine and its metabolization to putrescine allows for greater bacterial growth (in a biphasic pattern) and causes the alkalinization of the culture medium in a dose-dependent manner. The construction of a mutant lacking the AGDI cluster (L. lactis CECT 8666 Δagdi) confirmed the latter's direct role in putrescine production, growth, and medium alkalinization. Alkalinization did not affect the putrescine production pattern and was not essential for increased bacterial growth.

  13. Simultaneous HPTLC and RP-HPLC methods for determination of bumadizone in the presence of its alkaline-induced degradation product.

    Science.gov (United States)

    Ali, Nouruddin W; ZaaZaa, Hala A; Abdelkawy, M; Magdy, Maimana A

    2012-10-01

    Accurate, selective, sensitive and precise HPTLC-densitometric and RP-HPLC methods were developed and validated for determination of bumadizone calcium semi-hydrate in the presence of its alkaline-induced degradation product and in pharmaceutical formulation. Method A uses HPTLC-densitometry, depending on separation and quantitation of bumadizone and its alkaline-induced degradation product on TLC silica gel 60 F(254) plates, using hexane-ethyl acetate-glacial acetic acid (8:2:0.2, v/v/v) as a mobile phase followed by densitometric measurement of the bands at 240 nm. Method B comprises RP-HPLC separation of bumadizone and its alkaline-induced degradation product using a mobile phase consisting of methanol-water-acetonitrile (20:30:50, v/v/v) on a Phenomenex C(18) column at a flow-rate of 2 mL/min and UV detection at 235 nm. The proposed methods were successfully applied to the analysis of bumadizone either in bulk powder or in pharmaceutical formulation without interference from other dosage form additives, and the results were statistically compared with the established method.

  14. Multiresponse Optimization of Inoculum Conditions for the Production of Amylases and Proteases by Aspergillus awamori in Solid-State Fermentation of Babassu Cake

    Science.gov (United States)

    de Castro, Aline Machado; Teixeira, Mariana Martins Pereira; Carvalho, Daniele Fernandes; Freire, Denise Maria Guimarães; Castilho, Leda dos Reis

    2011-01-01

    This work aimed at investigating the simultaneous production of amylases and proteases by solid-state fermentation (SSF) of babassu cake using Aspergillus awamori IOC-3914. By means of experimental design techniques and the desirability function, optimum inoculum conditions (C/N ratio of propagation medium, inoculum age, and concentration of inoculum added to SSF medium) for the production of both groups of enzymes were found to be 25.8, 28.4 h, and 9.1 mg g−1, respectively. Significant influence of both initial C/N ratio and inoculum concentration was observed. Optimum amylolytic activities predicted by this multiresponse analysis were validated by independent experiments, thus indicating the efficacy of this approach. PMID:21915371

  15. Neutralization of acid mine drainage using the final product from CO{sub 2} emissions capture with alkaline paper mill waste

    Energy Technology Data Exchange (ETDEWEB)

    Perez-Lopez, Rafael, E-mail: rafael.perez@dgeo.uhu.es [Institute of Environmental Assessment and Water Research, IDAEA - CSIC, Jordi Girona 18, 08034 Barcelona (Spain); Department of Geology, University of Huelva, Campus ' El Carmen' , 21071, Huelva (Spain); Castillo, Julio; Quispe, Dino; Nieto, Jose Miguel [Department of Geology, University of Huelva, Campus ' El Carmen' , 21071, Huelva (Spain)

    2010-05-15

    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{sub 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{sup -1}, iron 348 mg L{sup -1}) with an average discharge of about 114 and 40 L s{sup -1} for calcium mud and calcite powder, respectively. Likewise, given the high potential of calcium mud to sequester CO{sub 2} and of resulting calcite powder to neutralize AMD, paper wastes could be a promising solution for facing this double environmental problem.

  16. 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...

  17. Mesoporous mordenites obtained by sequential acid and alkaline treatments – Catalysts for cumene production with enhanced accessibility

    NARCIS (Netherlands)

    van Laak, A.N.C.; Sagala, S.L.; Zecevic, J.; Friedrich, H.; de Jongh, P.E.; de Jong, K.P.

    2010-01-01

    Two commercially available mordenites, obtained from Zeolyst (Si/Al = 10 at/at) and BASF (Si/Al = 8 at/at), were subjected to post-synthesis treatments. The impact of acid treatment, alkaline treatment (desilication) and a combination of both on porosity, crystallinity and catalysis were studied in

  18. 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.

  19. 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

  20. Enhanced alkaline cellulases production by the thermohalophilic Aspergillus terreus AUMC 10138 mutated by physical and chemical mutagens using corn stover as substrate.

    Science.gov (United States)

    Isaac, George Saad; Abu-Tahon, Medhat Ahmed

    2015-01-01

    A thermohalophilic fungus, Aspergillus terreus AUMC 10138, isolated from the Wadi El-Natrun soda lakes in northern Egypt was exposed successively to gamma and UV-radiation (physical mutagens) and ethyl methan-sulfonate (EMS; chemical mutagen) to enhance alkaline cellulase production under solid state fermentation (SSF) conditions. The effects of different carbon sources, initial moisture, incubation temperature, initial pH, incubation period, inoculum levels and different concentrations of NaCl on production of alkaline filter paper activity (FPase), carboxymethyl cellulase (CMCase) and β-glucosidase by the wild-type and mutant strains of A. terreus were evaluated under SSF. The optimum conditions for maximum production of FPase, CMCase and β-glucosidase were found to be the corn stover: moisture ratio of 1:3(w/v), temperature 45 °C, pH range, 9.0-11.0, and fermentation for 4, 4 and 7 day, respectively. Inoculum levels of 30% for β-glucosidase and 40% for FPase, CMCase gave the higher cellulase production by the wild-type and mutant strains, respectively. Higher production of all three enzymes was obtained at a 5% NaCl. Under the optimized conditions, the mutant strain A. terreus M-17 produced FPase (729 U/g), CMCase (1,783 U/g), and β-glucosidase (342 U/g), which is, 1.85, 1.97 and 2.31-fold higher than the wild-type strain. Our results confirmed that mutant strain M-17 could be a promising alkaline cellulase enzyme producer employing lignocellulosics especially corn stover.

  1. 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.

  2. 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.

  3. Alkaline Hydrolysis/Polymerization of 2,4,6-Trinitrotoluene: Characterization of Products by 13C and 15N NMR

    Science.gov (United States)

    Thorn, K.A.; Thorne, P.G.; Cox, L.G.

    2004-01-01

    Alkaline hydrolysis has been investigated as a nonbiological procedure for the destruction of 2,4,6-trinitrotoluene (TNT) in explosives contaminated soils and munitions scrap. Nucleophilic substitutions of the nitro and methyl groups of TNT by hydroxide ion are the initial steps in the alkaline degradation of TNT. Potential applications of the technique include both in situ surface liming and ex situ alkaline treatment of contaminated soils. A number of laboratory studies have reported the formation of an uncharacterized polymeric material upon prolonged treatment of TNT in base. As part of an overall assessment of alkaline hydrolysis as a remediation technique, and to gain a better understanding of the chemical reactions underlying the hydrolysis/polymerization process, the soluble and precipitate fractions of polymeric material produced from the calcium hydroxide hydrolysis of unlabeled and 15N-labeled TNT were analyzed by elemental analysis and 13C and 15N nuclear magnetic resonance spectroscopy. Spectra indicated that reactions leading to polymerization included nucleophilic displacement of nitro groups by hydroxide ion, formation of ketone, carboxyl, alcohol, ether, and other aliphatic carbons, conversion of methyl groups to diphenyl methylene carbons, and recondensation of aromatic amines and reduced forms of nitrite, including ammonia and possibly hydroxylamine, into the polymer. Compared to the distribution of carbons in TNT as 14% sp 3- and 86% sp2-hybridized, the precipitate fraction from hydrolysis of unlabeled TNT contained 33% sp3- and 67% sp 2-hybridized carbons. The concentration of nitrogen in the precipitate was 64% of that in TNT. The 15N NMR spectra showed that, in addition to residual nitro groups, forms of nitrogen present in the filtrate and precipitate fractions include aminohydroquinone, primary amide, indole, imine, and azoxy, among others. Unreacted nitrite was recovered in the filtrate fraction. The toxicities and susceptibilities to

  4. Optimization of Fermentation Conditions for the Production of the M23 Protease Pseudoalterin by Deep-Sea Pseudoalteromonas sp. CF6-2 with Artery Powder as an Inducer

    Directory of Open Access Journals (Sweden)

    Hui-Lin Zhao

    2014-04-01

    Full Text Available Proteases in the M23 family have specific activities toward elastin and bacterial peptidoglycan. The peptidoglycan-degrading property makes these proteases have potential as novel antimicrobials. Because M23 proteases cannot be maturely expressed in Escherichia coli, it is significant to improve the production of these enzymes in their wild strains. Pseudoalterin is a new M23 protease secreted by the deep-sea bacterium Pseudoalteromonas sp. CF6-2. In this study, the fermentation conditions of strain CF6-2 for pseudoalterin production were optimized using single factor experiments and response surface methodology to improve the enzyme yield. To reduce the fermentation cost, bovine artery powder instead of elastin was determined as a cheap and efficient inducer. Based on single factor experiments, artery powder content, culture temperature and culture time were determined as the main factors influencing pseudoalterin production and were further optimized by the central composite design. The optimal values of these factors were determined as: artery powder of 1.2%, culture temperature of 20.17 °C and culture time of 28.04 h. Under the optimized conditions, pseudoalterin production reached 100.02 ± 9.0 U/mL, more than twice of that before optimization. These results lay a good foundation for developing the biotechnological potential of pseudoalterin.

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

    Science.gov (United States)

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

    2011-09-10

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

  6. 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.

  7. Essential role of mitogen-activated protein kinase pathways in protease activated receptor 2-mediated nitric-oxide production from rat primary astrocytes.

    Science.gov (United States)

    Park, Gyu Hwan; Jeon, Se Jin; Ryu, Jae Ryun; Choi, Min Sik; Han, Seol-Heui; Yang, Sung-Il; Ryu, Jong Hoon; Cheong, Jae Hoon; Shin, Chan Young; Ko, Kwang Ho

    2009-09-01

    Protease-activated receptors (PARs) play important roles in the regulation of brain function such as neuroinflammation by transmitting the signal from proteolytic enzymes such as thrombin and trypsin. We and others have reported that a member of the family, PAR-2 is activated by trypsin, whose involvement in the neurophysiological process is increasingly evident, and is involved in the neuroinflammatory processes including morphological changes of astrocytes. In this study, we investigated the role of PAR-2 in the production of nitric oxide (NO) in rat primary astrocytes. Treatment of PAR-2 agonist trypsin increased NO production in a dose-dependent manner, which was mediated by the induction of inducible nitric-oxide synthase. The trypsin-mediated production of NO was mimicked by PAR-2 agonist peptide and reduced by either pharmacological PAR-2 antagonist peptide or by siRNA-mediated inhibition of PAR-2 expression, which suggests the critical role of PAR-2 in this process. NO production by PAR-2 was mimicked by PMA, a PKC activator, and was attenuated by Go6976, a protein kinase C (PKC) inhibitor. PAR-2 stimulation activated three subtypes of mitogen-activated protein kinases (MAPKs): extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK. NO production by PAR-2 was blocked by inhibition of ERK, p38, and JNK pathways. PAR-2 stimulation also activated nuclear factor-kappaB (NF-kappaB) DNA binding and transcriptional activity as well as IkappaBalpha phosphorylation. Inhibitors of NF-kappaB pathway inhibited PAR-2-mediated NO production. In addition, inhibitors of MAPK pathways prevented transcriptional activation of NF-kappaB reporter constructs. These results suggest that PAR-2 activation-mediated NO production in astrocytes is transduced by the activation of MAPKs followed by NF-kappaB pathways.

  8. OPTIMIZATION OF MILK-CLOTTING PROTEASE PRODUCTION BY A LOCAL ISOLATE OF ASPERGILLUS NIGER FFB1 IN SOLID-STATE FERMENTATION

    Directory of Open Access Journals (Sweden)

    Souhila Bensmail

    2015-04-01

    Full Text Available The need to surmount the limitation of obtaining rennin, has been actively pushed researches to find new substitutes that present high milk-clotting activity which enables the production of high yields of cheese. In this study, the production of extracellular milk-clotting protease by locally isolated fungal specie, Aspergillus niger FFB1 under solid-state fermentation (SSF using cheep agro-industrial byproduct (wheat bran was optimized. The effects of several physicochemical and environmental factors were investigated to select the optimal conditions that ensure the best milk-clotting activity by application of "One-factor-at-a-time" method. A trial of cheese production using the crude extract was also carried out. The maximum enzyme activity (830 SU/g bran with a ratio MCA/PA of 4.25 was obtained under the optimum conditions of temperature (30°C, spores concentration (106 spores/mL, incubation time (72 hours, and moisture content of solid substrate (39.2% adjusted suitably with mineral solution (Czapek-Dox of pH 4.

  9. 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.

  10. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Watanabe, Kousuke; Emoto, Noriko; Sunohara, Mitsuhiro; Kawakami, Masanori; Kage, Hidenori; Nagase, Takahide; Ohishi, Nobuya [Department of Respiratory Medicine, The University of Tokyo Hospital, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-8655 (Japan); Takai, Daiya, E-mail: dtakai-ind@umin.ac.jp [Department of Clinical Laboratory, The University of Tokyo Hospital, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-8655 (Japan)

    2010-08-27

    Research highlights: {yields} Incubating PCR products at a high temperature causes smears in gel electrophoresis. {yields} Smears interfere with the interpretation of methylation analysis using COBRA. {yields} Treatment with exonuclease I and heat-labile alkaline phosphatase eliminates smears. {yields} 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 {sup o}C or 65 {sup 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.

  12. 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.

  13. 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.

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

    Institute of Scientific and Technical Information of China (English)

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

    2015-01-01

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

  15. Novel fungal protease.

    NARCIS (Netherlands)

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

    1994-01-01

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

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. Purification of a Factor from Human Placenta That Stimulates Capillary Endothelial Cell Protease Production, DNA Synthesis, and Migration

    Science.gov (United States)

    Moscatelli, David; Presta, Marco; Rifkin, Daniel B.

    1986-04-01

    A protein that stimulates the production of plasminogen activator and latent collagenase in cultured bovine capillary endothelial cells has been purified 106-fold from term human placenta by using a combination of heparin affinity chromatography, ion-exchange chromatography, and gel chromatography. The purified molecule has a molecular weight of 18,700 as determined by NaDodSO4/PAGE under both reducing and nonreducing conditions. The purified molecule stimulates the production of plasminogen activator and latent collagenase in a dose-dependent manner between 0.1 and 10 ng of protein/ml. The purified protein also stimulates DNA synthesis and chemotaxis in capillary endothelial cells in the same concentration range. Thus, this molecule has all of the properties predicted for an angiogenic factor.

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

    Science.gov (United States)

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

    2014-10-01

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

  3. Three genes expressing Kunitz domains in the epididymis are related to genes of WFDC-type protease inhibitors and semen coagulum proteins in spite of lacking similarity between their protein products

    Directory of Open Access Journals (Sweden)

    Lilja Hans

    2011-10-01

    Full Text Available Abstract Background We have previously identified a locus on human chromosome 20q13.1, encompassing related genes of postulated WFDC-type protease inhibitors and semen coagulum proteins. Three of the genes with WFDC motif also coded for the Kunitz-type protease inhibitor motif. In this report, we have reinvestigated the locus for homologous genes encoding Kunitz motif only. The identified genes have been analyzed with respect to structure, expression and function. Results We identified three novel genes; SPINT3, SPINT4 and SPINT5, and the structure of their transcripts were determined by sequencing of DNA generated by rapid amplification of cDNA ends. Each gene encodes a Kunitz domain preceded by a typical signal peptide sequence, which indicates that the proteins of 7.6, 8.7, and 9.7 kDa are secreted. Analysis of transcripts in 26 tissues showed that the genes predominantly are expressed in the epididymis. The recombinantly produced proteins could not inhibit the amidolytic activity of trypsin, chymotrypsin, plasmin, thrombin, coagulation factor Xa, elastase, urokinase and prostate specific antigen, whereas similarly made bovine pancreatic trypsin inhibitor (BPTI had the same bioactivity as the protein isolated from bovine pancreas. Conclusions The similar organization, chromosomal location and site of expression, suggests that the novel genes are homologous with the genes of WFDC-type protease inhibitors and semen coagulum proteins, despite the lack of similarity in primary structure of their protein products. Their restricted expression to the epididymis suggests that they could be important for male reproduction. The recombinantly produced proteins are presumably bioactive, as demonstrated with similarly made BPTI, but may have a narrower spectrum of inhibition, as indicated by the lacking activity against eight proteases with differing specificity. Another possibility is that they have lost the protease inhibiting properties, which is

  4. Production of Synthesis Gas via Methane Reforming with CO2 on Ni/SiO2 Catalysts Promoted by Alkali and Alkaline Earth Metals

    Institute of Scientific and Technical Information of China (English)

    陈平; 侯昭胤; 郑小明

    2005-01-01

    Ni/SiO2 catalysts promoted by alkali metals K and Cs or alkaline earth metals Mg, Ca, Sr and Ba were prepared, characterized by H2-TPR and XRD, and used for the production of synthesis gas via methane reforming with CO2. Though K and Cs promoted Ni catalysts could eliminate coke deposition, the reforming activity of these promoted catalysts was decreased heavily. Mg and Ca promoted Ni/SiO2 catalysts exhibited excellent coke resistance ability with minor loss of the reforming activity of Ni/SiO2. Ba showed poor coke resistance ability and small amount of Sr increased the formation of coke. The possible mechanism of these promoters was discussed.

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

    Institute of Scientific and Technical Information of China (English)

    Brian David Shaw; John Tane Christeller

    2009-01-01

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

  6. 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.

  7. Significantly enhancing recombinant alkaline amylase production in Bacillus subtilis by integration of a novel mutagenesis-screening strategy with systems-level fermentation optimization

    OpenAIRE

    2016-01-01

    Background Alkaline amylase has significant potential for applications in the textile, paper and detergent industries, however, low yield of which cannot meet the requirement of industrial application. In this work, a novel ARTP mutagenesis-screening method and fermentation optimization strategies were used to significantly improve the expression level of recombinant alkaline amylase in B. subtilis 168. Results The activity of alkaline amylase in mutant B. subtilis 168 mut-16# strain was 1.34...

  8. Effects of the use of ultrasonic waves on biodiesel production in alkaline transesterification of bleached tallow and vegetable oils: Cavitation model

    Science.gov (United States)

    Alape Benitez, Fabio

    Experiments of biodiesel production via methanolysis were performed at methanol/triglyceride molar ratios of 3, 4.5, and 6 and temperatures of 25°C, 40°C and 60°C; the reaction was monitored by HPLC, X-Ray, and GC-MS until equilibrium. A mathematical model called CAVITATION MODEL was developed to deal with mass transfer aspects of the alkaline transesterification reaction of vegetable oils; a comparison between the cavitation model and diffusion through spherical pores was made. Gas-vapor bubble dynamics for the methanol-soybean oil and methanol-tallow system were examined at 40°C and 42°C, respectively. The Rayleight-Plesset equations were used to describe the isothermal growth and adiabatic collapse of the bubble formed when a field of ultrasound at 20 KHz is applied. Temperatures of 2265 K and 426 K were estimated for a bubble in soybean oil-methanol and tallow-methanol systems, respectively. These "Hot Spots" could be responsible for the increment of the temperature occurred and the acoustic streaming observed during the alkaline transesterification reaction. Also, a diffusion analysis with the pore model was made to predict the concentration profile of the triglycerides within the liquid drops of alcohol created after the collapse of the gas-vapor bubbles; spherical shapes were studied. A computational model was made in MathCad to evaluate the effectiveness at different Thiele modulus values in order to estimate mass transfer coefficients for the most critical conditions of pure diffusion and these coefficients were compared with those found by the cavitation model estimation. Pictures of the reactant system soybean oil-methanol-potassium hydroxide, with the red dyed methanol using phenolphthalein, showed that the alkalinity of the system represented by potassium hydroxide remains in the interface alcohol-oil and then is displaced into the glycerol or down layer. The present study serves as a basis for the analysis of heterogeneous reactions with

  9. Bacterial proteases and virulence

    DEFF Research Database (Denmark)

    Frees, Dorte; Brøndsted, Lone; Ingmer, Hanne

    2013-01-01

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

  10. 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.

  11. [Use of coulometric titration for elucidating the mechanism of the oxidation of 6-APA alkaline breakdown products by halogens].

    Science.gov (United States)

    Kharlamov, V T; Inkin, A A; Ermolina, G E

    1975-02-01

    Penaldinic acid and penicillamine were formed on alkali decomposition (1 N NaOH) of 6-APA for 20 minutes at room temperature, penicillamine being completely oxidized to disulphide by the air oxygen. Coulometric titration of the alkali decomposition products showed that generated chlorine in 0.5 N HCl solution or bromine in a week acid solution of KBr oxidized them with participation of 7 electrones. Generated iodine did not practically oxidize the 6-APA decomposition products during the coulometric titration.

  12. Produção de proteases por Bacillus sp SMIA-2 crescido em soro de leite e água de maceração de milho e compatibilidade das enzimas com detergentes comerciais Production of proteases by Bacillus sp. SMIA-2 grow on whey and corn steep liquor and compatibility of the enzyme with commercial detergents

    Directory of Open Access Journals (Sweden)

    Wellingta Cristina Almeida do Nascimento

    2006-09-01

    Full Text Available A produção de proteases por Bacillus sp. SMIA-2 cultivado em um meio de cultura contendo soro de leite e água de maceração de milho foi estudada. Além disso, a compatibilidade da enzima com detergentes comerciais foi também avaliada. A atividade máxima da enzima (70 U/mg proteína foi observada na fase estacionária de crescimento, com 32 h de incubação. Estudos sobre a caracterização da protease revelaram que a temperatura ótima para atividade desta enzima foi 70 °C e que a mesma manteve 91% de sua atividade quando incubada a 70 °C na presença do cálcio. O valor ótimo de pH encontrado para a protease foi 8,0, sendo que a enzima manteve 85% e 46% de sua atividade quando incubada por 1 h em pH 9 e pH 10 respectivamente. A protease manteve 64% e 50% de sua atividade quando incubada a 70 °C por 30 min com os detergentes Cheer® e Tide® respectivamente. A utilização da glicina juntamente com íons cálcio resultou em um aumento da estabilidade enzimática em todos os detergentes testados. Em presença dos detergentes Ultra bizz®, Cheer® e Tide®, a enzima manteve aproximadamente 100% de atividade, após 30 min de incubação a 70 °C.The production of protease by the thermophilic Bacillus sp. SMIA-2 cultivated in a medium containing whey and corn steep liquor was studied. In addition, the compatibility of the enzyme with commercial detergents was evaluated. The maximum activity of the enzyme (70 U/mg protein was observed in the phase stationary of growth, with 32 h of incubation. Studies on the protease characterization revealed that the optimum temperature of this enzyme was 70 °C and that it maintained 91% of its activity when incubated a 70 °C in the presence of calcium. The optimum pH of the enzyme was found to be 8.0 and the enzyme maintained 85% and 46% of its original activity when incubated for 1 h at pH 9 and pH 10 respectively. Protease retained 64% and 50% of its activity after 30 min incubation at 70 °C in

  13. Determination of humic and fulvic acids in commercial solid and liquid humic products by alkaline extraction and gravimetric determination

    Science.gov (United States)

    Increased use of humic substances in agriculture has generated intense interest among producers, consumers, and regulators for an accurate and reliable method for quantification of humic (HA) and fulvic acids (FA) in raw ores and products. Here we present a thoroughly validated method, the Humic Pro...

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

    Science.gov (United States)

    Zółtowska, Krystyna; Lipiński, Zbigniew; Dmitryjuk, Małgorzata

    2005-01-01

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

  15. 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, pmethane 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.

  16. 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.

    Science.gov (United States)

    Tantis, Iosif; Bousiakou, Leda; Frontistis, Zacharias; Mantzavinos, Dionissios; Konstantinou, Ioannis; Antonopoulou, Maria; Karikas, George-Albert; Lianos, Panagiotis

    2015-08-30

    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(-4)min(-1) under low intensity UVA irradiation of 1.5mWcm(-2) in the presence of a nanoparticulate titania film. This corresponds to degradation of 1.4mg 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(-4)min(-1) by applying a forward bias of +0.6V 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.

  17. Construction of Aspergillus niger integrated with cellulase gene from Ampullaria gigas Spix for improved enzyme production and saccharification of alkaline-pretreated rice straw.

    Science.gov (United States)

    Yang, Peizhou; Zhang, Haifeng; Cao, Lili; Zheng, Zhi; Jiang, Shaotong

    2016-12-01

    Aspergillus niger is an important microorganism that has been used for decades to produce extracellular enzymes. In this study, a novel Aspergillus niger strain integrated with a eukaryotic expression vector harboring the gpd-Shi promoter of shiitake mushrooms and cellulase gene of Ampullaria gigas Spix was engineered to improve cellulase production for the achievement of highly efficient saccharification of agricultural residues. In one strain, designated ACShi27, which exhibited the highest total cellulase expression, total cellulase, endoglucanase, exoglucanase, and xylanase expression levels were 1.73, 16.23, 17.73, and 150.83 U ml(-1), respectively; these values were 14.5, 22.3, 24.6, and 17.3% higher than those of the wild-type Aspergillus niger M85 using wheat bran as an induction substrate. Production of cellulases and xylanase by solid-state fermentation followed by in situ saccharification of ACShi27 was investigated with alkaline-pretreated rice straw as a substrate. After 2 days of enzyme induction at 30 °C, followed by 48 h of saccharification at 50 °C, the conversion rate of carbon polymers into reducing sugar reached 293.2 mg g(-1), which was 1.23-fold higher than that of the wild-type strain. The expression of sestc in Aspergillus niger can improve the total cellulase and xylanase activity and synergism, thereby enhancing the lignocellulose in situ saccharification.

  18. Peptide synthesis in neat organic solvents with novel thermostable proteases

    NARCIS (Netherlands)

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

    2015-01-01

    Biocatalytic peptide synthesis will benefit from enzymes that are active at low water levels in organic solvent compositions that allow good substrate and product solubility. To explore the use of proteases from thermophiles for peptide synthesis under such conditions, putative protease genes of the

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

    Institute of Scientific and Technical Information of China (English)

    刘芳; 曹新志; 游见明; 刘春明

    2012-01-01

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

  20. 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

  1. 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.

  2. Production, properties and application to biocatalysis of a novel extracellular alkaline phenol oxidase from the thermophilic fungus Scytalidium thermophilum.

    Science.gov (United States)

    Ogel, Z B; Yüzügüllü, Y; Mete, S; Bakir, U; Kaptan, Y; Sutay, D; Demir, A S

    2006-08-01

    Scytalidium thermophilum produces an extracellular phenol oxidase on glucose-containing medium. Certain phenolic acids, specifically gallic acid and tannic acid, induce the expression of the enzyme. Production at 45 degrees C in batch cultures is growth-associated and is enhanced in the presence of 160 microM CuSO4 x 5 H2O and 3 mM gallic acid. The highest enzyme activity is observed at pH 7.5 and 65 degrees C, on catechol. When incubated for 1 h at pH 7 and pH 8, 95% and 86% of the activity is retained. Thermostability decreases gradually from 40 degrees C to 80 degrees C. Estimated molecular mass is c. 83 kDa, and pI is acidic at c. 5.4. Substrate specificity and inhibition analysis in culture supernatants suggest that the enzyme has unique properties showing activity towards catechol; 3,4-dihydroxy-L-phenylalanine (L-DOPA); 4-amino-N, N-diethylaniline (ADA); p-hydroquinone; gallic acid; tannic acid and caffeic acid, and no activity towards L-tyrosine, guaiacol, 2,2'-azino-bis(3-ethyl-benzthiazoline-6-sulphonic acid) (ABTS) and syringaldazine. Inhibition is observed in the presence of salicyl hydroxamic acid (SHAM) and p-coumaric acid. Enzyme activity is enhanced by cetyltrimethylammonium bromide (CTAB) and polyvinylpyrrolidone (PVP), and the organic solvents dimethyl sulfoxide (DMSO) and ethanol. No inhibition is observed in the presence of carbon monoxide. Benzoin, benzoyl benzoin and hydrobenzoin are converted into benzil, and stereoselective oxidation is observed on hydrobenzoin. The reported enzyme is novel due to its catalytic properties resembling mainly catechol oxidases, but displaying some features of laccases at the same time.

  3. 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

  4. Death proteases come alive

    NARCIS (Netherlands)

    Woltering, E.J.

    2004-01-01

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

  5. Cold-adapted proteases as an emerging class of therapeutics.

    Science.gov (United States)

    Fornbacke, Marcus; Clarsund, Mats

    2013-06-01

    Proteases have been used in medicine for several decades and are an established and well tolerated class of therapeutic agent. These proteases were sourced from mammals or bacteria that exist or have adapted to moderate temperatures (mesophilic organisms); however, proteases derived from organisms from cold environments-cold-adapted or psychrophilic proteases-generally have high specific activity, low substrate affinity, and high catalytic rates at low and moderate temperatures. Made possible by greater flexibility, psychrophilic enzymes interact with and transform the substrate at lower energy costs. Cold-adapted proteases have been used in a wide range of applications, including industrial functions, textiles, cleaning/hygiene products, molecular biology, environmental bioremediations, consumer food products, cosmetics, and pharmaceutical production. In addition to these applications, they have also shown promise as therapeutic modalities for cosmeceutical applications (by reducing glabellar [frown] lines) and a number of disease conditions, including bacterial infections (by disrupting biofilms to prevent bacterial infection), topical wound management (when used as a debridement agent to remove necrotic tissue and fibrin clots), oral/dental health management (by removing plaque and preventing periodontal disease), and in viral infections (by reducing the infectivity of viruses, such as human rhinovirus 16 and herpes simplex virus). Psychrophilic proteases with greater activity and stability (than the original organism-derived variant) have been developed; this coupled with available manufacturing recombinant production techniques suggests that cold-adapted proteases have a promising future as a distinct therapeutic class with diverse clinical applications.

  6. 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......) constitutes an excellent adsorptive method for efficient extracellular protease removal during cultivation. In this work, the impact of semi‐continuous ISMS on the overall protease yield has been investigated. Results reveal significant removal of the protease from Bacillus licheniformis cultivations....... Bacitracin‐functionalized magnetic particles were successfully applied, regenerated and reused up to 30 times. Immediate reproduction of the protease after ISMS proved the biocompatibility of this integrated approach. Six subsequent ISMS steps significantly increased the overall protease yield up to 98...

  7. Anodes for alkaline electrolysis

    Science.gov (United States)

    Soloveichik, Grigorii Lev

    2011-02-01

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

  8. 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.

  9. Determination of a novel ACE inhibitor in the presence of alkaline and oxidative degradation products using smart spectrophotometric and chemometric methods

    Institute of Scientific and Technical Information of China (English)

    Maha Abdel-Monem Hegazy; Maya Shaaban Eissa; Osama Ibrahim Abd El-Sattar; Mohamed Mohamed Abd El-Kawy

    2014-01-01

    Simple, accurate, sensitive and validated UV spectrophotometric and chemometric methods were developed for the determination of imidapril hydrochloride (IMD) in the presence of both its alkaline (AKN) and oxidative (OXI) degradation products and in its pharmaceutical formulation. Method A is the fourth derivative spectra (D4) which allows the determination of IMD in the presence of both AKN and OXD, in pure form and in tablets by measuring the peak amplitude at 243.0 nm. Methods B, C and D, manipulating ratio spectra, were also developed. Method B is the double divisor-ratio difference spectrophotometric one (DD-RD) by computing the difference between the amplitudes of IMD ratio spectra at 232 and 256.3 nm. Method C is the double divisor-first derivative of ratio spectra method (DD-DR1) at 243.2 nm, while method D is the mean centering of ratio spectra (MCR) at 288.0 nm. Methods A, B, C and D could successfully determine IMD in a concentration range of 4.0-32.0 mg/mL. Methods E and F are principal component regression (PCR) and partial least-squares (PLS), respectively, for the simultaneous determination of IMD in the presence of both AKN and OXI, in pure form and in its tablets. The developed methods have the advantage of simultaneous determination of the cited components without any pre-treatment. The accuracy, precision and linearity ranges of the developed methods were determined. The results obtained were statistically compared with those of a reported HPLC method, and there was no significant difference between the proposed methods and the reported method regarding both accuracy and precision.

  10. Proteases, neutrophils, and periodontitis: the NET effect.

    Science.gov (United States)

    Nauseef, William M

    2014-10-01

    Neutrophils exert potent antimicrobial activities in their role as first-line cellular defenders against infection. The synergistic and collective actions of oxidants and granule proteins, including serine proteases, support the microbial killing in phagosomes, where most neutrophil-mediated antimicrobial action occurs. In addition to phagocytosis, specific stimuli prompt neutrophils to extrude a matrix of DNA, histones, and granule proteins to produce neutrophil extracellular traps (NETs), which can trap microbes. Mice lacking the serine proteases necessary for NET production are more susceptible to infection, an observation suggesting that functional NETs are required for host protection. In this issue of the JCI, Sørensen and colleagues characterize neutrophils from a patient with Papillon-Lefèvre syndrome. The patient has an inactivating mutation in the gene encoding dipeptidyl peptidase I, resulting in neutrophils lacking elastase, a serine protease required for NET production. Despite the inability to form NETS, neutrophils from this patient killed pathogens in vitro, and the patient did not exhibit evidence of an increased propensity toward bacterial infections. Together, these results suggest that proteases in human neutrophils are dispensable for protection against bacterial infection and that the ability to generate NETs in vitro does not compromise host defense.

  11. From proteases to proteomics.

    Science.gov (United States)

    Neurath, H

    2001-04-01

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

  12. Alkaline battery operational methodology

    Science.gov (United States)

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

    2016-08-16

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

  13. Functional expression of a novel alkaline-adapted lipase of Bacillus amyloliquefaciens from stinky tofu brine and development of immobilized enzyme for biodiesel production.

    Science.gov (United States)

    Cai, Xianghai; Ma, Jing; Wei, Dong-Zhi; Lin, Jin-Ping; Wei, Wei

    2014-11-01

    Using enrichment procedures, a lipolytic strain was isolated from a stinky tofu brine and was identified as Bacillus amyloliquefaciens (named B. amyloliquefaciens Nsic-8) by morphological, physiological, biochemical tests and 16S rDNA sequence analysis. Meanwhile, the key enzyme gene (named lip BA) involved in ester metabolism was obtained from Nsic-8 with the assistance of homology analysis. The novel gene has an open reading frame of 645 bp, and encodes a 214-amino-acid lipase (LipBA). The deduced amino acid sequence shows the highest identity with the lipase from B. amyloliquefaciens IT-45 (NCBI database) and belongs to the family of triacylglycerol lipase (EC 3.1.1.3). The lipase gene was expressed in Escherichia coli BL21(DE3) using plasmid pET-28a. The enzyme activity and specific activity were 250 ± 16 U/ml and 1750 ± 153 U/mg, respectively. The optimum pH and temperature of the recombinant enzyme were 9.0 and 40 °C respectively. LipBA showed much higher stability under alkaline conditions and was stable at pH 7.0-11.0. The Km and Vmax values of purified LipBA using 4-nitrophenyl palmitate as the substrate were 1.04 ± 0.06 mM and 119.05 ± 7.16 μmol/(ml min), respectively. After purification, recombinant lipase was immobilized with the optimal conditions (immobilization time 3 h at 30 °C, with 92 % enzyme recovery) and the immobilized enzyme was applied in biodiesel production. This is the first report of the lipase activity and lipase gene obtained from B. amyloliquefaciens (including wild strain and recombinant strain) and the recombinant LipBA with the detailed enzymatic properties. Also the preliminary study of the transesterification shows the potential value in biodiesel production applications.

  14. Production of bioactive peptide hydrolysates from deer, sheep, pig and cattle red blood cell fractions using plant and fungal protease preparations.

    Science.gov (United States)

    Bah, Clara S F; Carne, Alan; McConnell, Michelle A; Mros, Sonya; Bekhit, Alaa El-Din A

    2016-07-01

    Protease preparations from plant (papain and bromelain) and fungal (FP400 and FPII) sources were used to hydrolyze the red blood cell fractions (RBCFs) separated from deer, sheep, pig, and cattle abattoir-sourced blood. After 1, 2, 4 and 24h of hydrolysis, the antioxidant and antibacterial activities of the peptide hydrolysates obtained were investigated. The increase in trichloroacetic acid-soluble peptides over the hydrolysis period was examined using the o-phthaldialdehyde (OPA) assay and the hydrolysis profiles were illustrated using SDS-PAGE. Papain generated RBCF hydrolysates exhibited higher ferric reducing antioxidant power (FRAP) and oxygen radical absorbance capacity (ORAC) compared to those generated with bromelain, FP400 and FPII. At certain concentrations, 24h hydrolysates of RBCF using FP400 and FPII were able to inhibit the growth of Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa. The results indicated that the use of proteases from plant or fungal sources can produce animal blood hydrolysates with antioxidant and antimicrobial activities.

  15. Cell-free production of integral membrane aspartic acid proteases reveals zinc-dependent methyltransferase activity of the Pseudomonas aeruginosa prepilin peptidase PilD

    Science.gov (United States)

    Aly, Khaled A; Beebe, Emily T; Chan, Chi H; Goren, Michael A; Sepúlveda, Carolina; Makino, Shin-ichi; Fox, Brian G; Forest, Katrina T

    2013-01-01

    Integral membrane aspartic acid proteases are receiving growing recognition for their fundamental roles in cellular physiology of eukaryotes and prokaryotes, and may be medically important pharmaceutical targets. The Gram-negative Pseudomonas aeruginosa PilD and the archaeal Methanococcus voltae FlaK were synthesized in the presence of unilamellar liposomes in a cell-free translation system. Cosynthesis of PilD with its full-length substrate, PilA, or of FlaK with its full-length substrate, FlaB2, led to complete cleavage of the substrate signal peptides. Scaled-up synthesis of PilD, followed by solubilization in dodecyl-β-d-maltoside and chromatography, led to a pure enzyme that retained both of its known biochemical activities: cleavage of the PilA signal peptide and S-adenosyl methionine-dependent methylation of the mature pilin. X-ray fluorescence scans show for the first time that PilD is a zinc-binding protein. Zinc is required for the N-terminal methylation of the mature pilin, but not for signal peptide cleavage. Taken together, our work identifies the P. aeruginosa prepilin peptidase PilD as a zinc-dependent N-methyltransferase and provides a new platform for large-scale synthesis of PilD and other integral membrane proteases important for basic microbial physiology and virulence. PMID:23255525

  16. 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.

  17. 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

  18. Nucleic Acid Aptamers Against Proteases

    DEFF Research Database (Denmark)

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

    2011-01-01

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

  19. Cathepsin proteases in Toxoplasma gondii

    OpenAIRE

    Dou, Zhicheng; Carruthers, Vern B.

    2011-01-01

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

  20. PROTEASES AND PROTEASE INHIBITORS INTERACTION: DEFENCE STRATEGY AGAINST

    Directory of Open Access Journals (Sweden)

    R.S.DHANDE 1 N.J.CHIKHALE 2

    2014-12-01

    Full Text Available ABSTRACT: An increase in crop yield, its management and preservation are among the main challenges standing before the human population that exceed 10 billion by the mid of 21 st  century.  Every year, considerable agricultural losses occur due to repeated practices of cultivation of large genetically similar populations.  Such cultivation practices favors incidence of more insect pests (Hilder and Boulter, 1999;  Oerke  et  al.,  1994;  Smith,  1999.  To  solve  these  problems,  current approaches  rely  on  use  of  synthetic  chemicals  like  fertilizers,  insecticides, herbicides,  fungicides  etc.  But  this  exerts  excessively  high  pressure  on environment  and  destabilizes  the  ecological  balance.  The  traditional  pest control method involves the use of conventional pesticides, most of which are non-specific and wipe out the entire community, pollutes the agro-ecosystem, and  increases  the  cost  of  production.  The  emergence  of  gene  transfer technology  has  solved  some  problems  regarding  overuse  of  chemical pesticides.  The  delta  endotoxin  encoding  gene  from  Bacillus  thuringiensis,  a gram positive soil borne bacteria transferred in crops has given little relief from coleopterans and lepidopterans attack.  Whereas, the insects belonging to these orders like Helicoverpa Sps. have developed resistance against Bt toxins. The other approach takes advantage of use of plant genes encoding defense proteins like protease inhibitors which is more appealing, simpler and safer (Dunaevsky et.  al.,  2005.  Proteinase  inhibitors  (PIs  are  naturally  occurring  proteins  in living organisms and are able to inhibit & control the activity of proteases. PIs act  on  an  active  site  of  digestive  proteolytic  enzymes  and  form  a  stable complex  unlike  enzyme-substrate  or  enzyme-product  weak  complexes  which

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

    Directory of Open Access Journals (Sweden)

    Manabe Kenji

    2012-06-01

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

  2. Protease-mediated drug delivery

    Science.gov (United States)

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

    2003-12-01

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

  3. Proteases of Wood Rot Fungi with Emphasis on the Genus Pleurotus

    Directory of Open Access Journals (Sweden)

    Fabíola Dorneles Inácio

    2015-01-01

    Full Text Available Proteases are present in all living organisms and they play an important role in physiological conditions. Cell growth and death, blood clotting, and immune defense are all examples of the importance of proteases in maintaining homeostasis. There is growing interest in proteases due to their use for industrial purposes. The search for proteases with specific characteristics is designed to reduce production costs and to find suitable properties for certain industrial sectors, as well as good producing organisms. Ninety percent of commercialized proteases are obtained from microbial sources and proteases from macromycetes have recently gained prominence in the search for new enzymes with specific characteristics. The production of proteases from saprophytic basidiomycetes has led to the identification of various classes of proteases. The genus Pleurotus has been extensively studied because of its ligninolytic enzymes. The characteristics of this genus are easy cultivation techniques, high yield, low nutrient requirements, and excellent adaptation. There are few studies in the literature about proteases of Pleurotus spp. This review gathers together information about proteases, especially those derived from basidiomycetes, and aims at stimulating further research about fungal proteases because of their physiological importance and their application in various industries such as biotechnology and medicine.

  4. 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

    Feed industries are seeking new ways to cope with increased raw material costs, and one approach is to apply enzymatic treatment in the production of feed ingredients from animal by-products. Keratinases, a group of proteases, are capable of hydrolyzing keratin-rich material and have been applied...... such as dithiothreitol (DTT) and Na2SO3. In general, the protease from B. subtilis was more efficient in degrading feather keratin compared to the other 3 feed proteases at both pH 5.5 and 7.0. For commercial production, the application of protease from B. subtilis is even more advantageous considering the lower cost-in-use....

  5. Alkaline Hydrolysis/Polymerization of 2,4,6-Trinitrotoluene:  Characterization of Products by 13C and 15N NMR

    Science.gov (United States)

    Thorn, Kevin A.; Thorne, Philip G.; Cox, Larry G.

    2004-01-01

    Alkaline hydrolysis has been investigated as a nonbiological procedure for the destruction of 2,4,6-trinitrotoluene (TNT) in explosives contaminated soils and munitions scrap. Nucleophilic substitutions of the nitro and methyl groups of TNT by hydroxide ion are the initial steps in the alkaline degradation of TNT. Potential applications of the technique include both in situ surface liming and ex situ alkaline treatment of contaminated soils. A number of laboratory studies have reported the formation of an uncharacterized polymeric material upon prolonged treatment of TNT in base. As part of an overall assessment of alkaline hydrolysis as a remediation technique, and to gain a better understanding of the chemical reactions underlying the hydrolysis/polymerization process, the soluble and precipitate fractions of polymeric material produced from the calcium hydroxide hydrolysis of unlabeled and 15N-labeled TNT were analyzed by elemental analysis and 13C and 15N nuclear magnetic resonance spectroscopy. Spectra indicated that reactions leading to polymerization included nucleophilic displacement of nitro groups by hydroxide ion, formation of ketone, carboxyl, alcohol, ether, and other aliphatic carbons, conversion of methyl groups to diphenyl methylene carbons, and recondensation of aromatic amines and reduced forms of nitrite, including ammonia and possibly hydroxylamine, into the polymer. Compared to the distribution of carbons in TNT as 14% sp3- and 86% sp2-hybridized, the precipitate fraction from hydrolysis of unlabeled TNT contained 33% sp3- and 67% sp2-hybridized carbons. The concentration of nitrogen in the precipitate was 64% of that in TNT. The 15N NMR spectra showed that, in addition to residual nitro groups, forms of nitrogen present in the filtrate and precipitate fractions include aminohydroquinone, primary amide, indole, imine, and azoxy, among others. Unreacted nitrite was recovered in the filtrate fraction. The toxicities and susceptibilities to

  6. Characterization and expression levels of protease enzymes at different developmental stages of Siamese fighting fish (Betta splendens Regan, 1910)

    OpenAIRE

    Thongprajukaew, Karun; Kovitvadhi, Uthaiwan; Engkagul, Arunee; Torrissen, Krisna Rungruangsak

    2010-01-01

    Characterization of total proteases, trypsin and chymotrypsin were performed at pH 2 12 and temperatures 20-80°C in three life stages of Siamese fighting fish (Betta splendens Regan, 1910). At least fourteen protease activities were detected, including seven acidic (pH levels 2, 4 and 5-6), one neutral (pH 7), and six alkaline (pH levels 8-12) activities in the optimal temperature range of 30-60°C. The neutral and alkaline pH profiles were different between sexes during the on-gro...

  7. Alkaline earth metal thioindates

    Energy Technology Data Exchange (ETDEWEB)

    Ivanov-Ehmin, B.N.; Ivlieva, V.I.; Filatenko, L.A.; Zajtsev, B.E.; Kaziev, G.Z.; Sarabiya, M.G.

    1984-08-01

    Alkaline earth metal thioindates of MIn/sub 2/S/sub 4/ composition were synthesized by interaction of alkaline earth metal oxoindates with hydrogen sulfide during heating. Investigation into the compounds by X-ray analysis showed that calcium compound crystallizes in cubic crystal system and strontium and barium compounds in rhombic crystal system. Lattice parameters and the number of formula units were determined. Thioindates of M/sub 3/In/sub 2/S/sub 6/ composition were synthesized, their individuality was shown.

  8. Optimization of CO{sub 2} use for cores and molds production using phenolic alkaline resin; Otimizacao do uso do CO{sub 2} na fabricacao de machos e moldes, utilizando resina fenolica alcalina gasada

    Energy Technology Data Exchange (ETDEWEB)

    Del Negro Sutter, Luiz Edmundo; Egreja, Jose Amilcar; Andrade, Jose Espedito de; Doroteu Neto, Antonio

    1996-12-31

    This technical paper describes the experiments held in lab scale with objective of optimizing CO{sub 2} gassing in the phenolic alkaline process. This paper also emphasizes the influence and the concern of the pressure and gassing time, CO{sub 2} temperature and flow rate control in order to obtain a complete hardening of the core and mould. After these experiments, and additional analysis was held in order to evaluate the quality of the casting as compared to the traditional process for cores and molds production. (author) 3 refs., 11 figs., 16 tabs.

  9. Proteases induce secretion of collagenase and plasminogen activator by fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Werb, Z.; Aggeler, J.

    1978-04-01

    We have observed that treatment of rabbit synovial fibroblasts with proteolytic enzymes can induce secretion of collagenase (EC 3.4.24.7) and plasminogen activator (EC 3.4.21.-). Cells treated for 2 to 24 hr with plasmin, trypsin, chymotrypsin, pancreatic elastase, papain, bromelain, thermolysin, or ..cap alpha..-protease but not with thrombin or neuraminidase secreted detectable amounts of collagenase within 16 to 48 hr. Treatment of fibroblasts with trypsin also induced secretion of plasminogen activator. Proteases initiated secretion of collagenase (up to 20 units per 10/sup 6/ cells per 24 hr) only when treatment produced decreased cell adhesion. Collagenase production did not depend on continued presence of proteolytic activity or on subsequent cell adhesion, spreading, or proliferation. Routine subculturing with crude trypsin also induced collagenase secretion by cells. Secretion of collagenase was prevented and normal spreading was obtained if the trypsinized cells were placed into medium containing fetal calf serum. Soybean trypsin inhibitor, ..cap alpha../sub 1/-antitrypsin, bovine serum albumin, collagen, and fibronectin did not inhibit collagenase production. Although proteases that induced collagenase secretion also removed surface glycoprotein, the kinetics of induction of cell protease secretion were different from those for removal of fibronectin. Physiological inducers of secretion of collagenase and plasminogen activator by cells have not been identified. These results suggest that extracellular proteases in conjunction with plasma proteins may govern protease secretion by cells.

  10. The effect of environmental conditions on extracellular protease activity in controlled fermentations of Aspergillus niger

    NARCIS (Netherlands)

    Braaksma, M.; Smilde, A.K.; Werf, M.J. van der; Punt, P.J.

    2009-01-01

    Proteolytic degradation by host proteases is one of the key issues in the application of filamentous fungi for non-fungal protein production. In this study the influence of several environmental factors on the production of extracellular proteases of Aspergillus niger was investigated systematically

  11. 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.

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

    Science.gov (United States)

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

    2014-01-01

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

  13. Cathepsin proteases in Toxoplasma gondii

    Science.gov (United States)

    Dou, Zhicheng; Carruthers, Vern B.

    2014-01-01

    Cysteine proteases are important for the growth and survival of apicomplexan parasites that infect humans. The apicomplexan Toxoplasma gondii expresses five members of the C1 family of cysteine proteases, including one cathepsin L-like (TgCPL), one cathepsin B-like (TgCPB), and three cathepsin C-like (TgCPC1, 2 and 3) proteases. Recent genetic, biochemical and structural studies reveal that cathepsins function in microneme and rhoptry protein maturation, host cell invasion, replication, and nutrient acquisition.. Here, we review the key features and roles of T. gondii cathepsins and discuss the therapeutic potential for specific inhibitor development. PMID:21660658

  14. Genome-wide survey of prokaryotic serine proteases: Analysis of distribution and domain architectures of five serine protease families in prokaryotes

    Directory of Open Access Journals (Sweden)

    Tripathi Lokesh P

    2008-11-01

    Full Text Available Abstract Background Serine proteases are one of the most abundant groups of proteolytic enzymes found in all the kingdoms of life. While studies have established significant roles for many prokaryotic serine proteases in several physiological processes, such as those associated with metabolism, cell signalling, defense response and development, functional associations for a large number of prokaryotic serine proteases are relatively unknown. Current analysis is aimed at understanding the distribution and probable biological functions of the select serine proteases encoded in representative prokaryotic organisms. Results A total of 966 putative serine proteases, belonging to five families, were identified in the 91 prokaryotic genomes using various sensitive sequence search techniques. Phylogenetic analysis reveals several species-specific clusters of serine proteases suggesting their possible involvement in organism-specific functions. Atypical phylogenetic associations suggest an important role for lateral gene transfer events in facilitating the widespread distribution of the serine proteases in the prokaryotes. Domain organisations of the gene products were analysed, employing sensitive sequence search methods, to infer their probable biological functions. Trypsin, subtilisin and Lon protease families account for a significant proportion of the multi-domain representatives, while the D-Ala-D-Ala carboxypeptidase and the Clp protease families are mostly single-domain polypeptides in prokaryotes. Regulatory domains for protein interaction, signalling, pathogenesis, cell adhesion etc. were found tethered to the serine protease domains. Some domain combinations (such as S1-PDZ; LON-AAA-S16 etc. were found to be widespread in the prokaryotic lineages suggesting a critical role in prokaryotes. Conclusion Domain architectures of many serine proteases and their homologues identified in prokaryotes are very different from those observed in eukaryotes

  15. Metal-based antimicrobial protease inhibitors.

    Science.gov (United States)

    Kellett, A; Prisecaru, A; Slator, C; Molphy, Z; McCann, M

    2013-01-01

    Limitations associated with the production cost, metabolic instability, side-effects, resistance and poor pharmacokinetics of organic protease inhibitors (PIs), which form an essential component of the front line HAART treatment for HIV, have fuelled efforts into finding novel, transition metal-based alternatives. Some of the attractive features of metalbased therapeutics include synthetic simplicity, solubility control, redox capability, expansion of coordination number and topography matching of the complex to the protein's active site. Building asymmetry into the complex, which may offer better discrimination between host and rogue cell, can readily be achieved through coordination of chiral ligands to the metal centre. Although the scope of this review has been limited to metal-based agents that have been reported to bind/inhibit HIV-1 and parasitic proteases, some desirables, such as high activity, low dosage, minimal toxicity, crossinhibition, unique binding modes and selectivity, have already been delivered. The variability of the d-block metals, coupled with the availability of designer organic ligands, augers well for the future development of clinical metallo-drugs for deployment against protease-associated, fatal diseases.

  16. 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.

  17. A computational module assembled from different protease family motifs identifies PI PLC from Bacillus cereus as a putative prolyl peptidase with a serine protease scaffold.

    Science.gov (United States)

    Rendón-Ramírez, Adela; Shukla, Manish; Oda, Masataka; Chakraborty, Sandeep; Minda, Renu; Dandekar, Abhaya M; Ásgeirsson, Bjarni; Goñi, Félix M; Rao, Basuthkar J

    2013-01-01

    Proteolytic enzymes have evolved several mechanisms to cleave peptide bonds. These distinct types have been systematically categorized in the MEROPS database. While a BLAST search on these proteases identifies homologous proteins, sequence alignment methods often fail to identify relationships arising from convergent evolution, exon shuffling, and modular reuse of catalytic units. We have previously established a computational method to detect functions in proteins based on the spatial and electrostatic properties of the catalytic residues (CLASP). CLASP identified a promiscuous serine protease scaffold in alkaline phosphatases (AP) and a scaffold recognizing a β-lactam (imipenem) in a cold-active Vibrio AP. Subsequently, we defined a methodology to quantify promiscuous activities in a wide range of proteins. Here, we assemble a module which encapsulates the multifarious motifs used by protease families listed in the MEROPS database. Since APs and proteases are an integral component of outer membrane vesicles (OMV), we sought to query other OMV proteins, like phospholipase C (PLC), using this search module. Our analysis indicated that phosphoinositide-specific PLC from Bacillus cereus is a serine protease. This was validated by protease assays, mass spectrometry and by inhibition of the native phospholipase activity of PI-PLC by the well-known serine protease inhibitor AEBSF (IC50 = 0.018 mM). Edman degradation analysis linked the specificity of the protease activity to a proline in the amino terminal, suggesting that the PI-PLC is a prolyl peptidase. Thus, we propose a computational method of extending protein families based on the spatial and electrostatic congruence of active site residues.

  18. 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

  19. Alkaline broadening in Stars

    CERN Document Server

    De Kertanguy, A

    2015-01-01

    Giving new insight for line broadening theory for atoms with more structure than hydrogen in most stars. Using symbolic software to build precise wave functions corrected for ds;dp quantum defects. The profiles obtained with that approach, have peculiar trends, narrower than hydrogen, all quantum defects used are taken from atomic database topbase. Illustration of stronger effects of ions and electrons on the alkaline profiles, than neutral-neutral collision mechanism. Keywords : Stars: fundamental parameters - Atomic processes - Line: profiles.

  20. Increased river alkalinization in the Eastern U.S

    Science.gov (United States)

    Kaushal, S.; Likens, G. E.; Utz, R.; Pace, M.; Grese, M.; Yepsen, M.

    2013-12-01

    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 km2. 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 3 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.

  1. 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.

  2. Use of the Protease Fluorescent Detection Kit to Determine Protease Activity

    OpenAIRE

    Cupp-Enyard, Carrie

    2009-01-01

    The Protease Fluorescent Detection Kit provides ready-to-use reagents for detecting the presence of protease activity. This simple assay to detect protease activity uses casein labeled with fluorescein isothiocyanate (FITC) as the substrate.

  3. Application of a cell-based protease assay for testing inhibitors of picornavirus 3C proteases

    NARCIS (Netherlands)

    van der Linden, Lonneke; Ulferts, Rachel; Nabuurs, Sander B; Kusov, Yuri; Liu, Hong; George, Shyla; Lacroix, Céline; Goris, Nesya; Lefebvre, David; Lanke, Kjerstin H W; De Clercq, Kris; Hilgenfeld, Rolf; Neyts, Johan; van Kuppeveld, Frank J M

    2014-01-01

    Proteolytical cleavage of the picornaviral polyprotein is essential for viral replication. Therefore, viral proteases are attractive targets for anti-viral therapy. Most assays available for testing proteolytical activity of proteases are performed in vitro, using heterologously expressed proteases

  4. Serine proteases, serine protease inhibitors, and protease-activated receptors: roles in synaptic function and behavior.

    Science.gov (United States)

    Almonte, Antoine G; Sweatt, J David

    2011-08-17

    Serine proteases, serine protease inhibitors, and protease-activated receptors have been intensively investigated in the periphery and their roles in a wide range of processes-coagulation, inflammation, and digestion, for example-have been well characterized (see Coughlin, 2000; Macfarlane et al., 2001; Molinari et al., 2003; Wang et al., 2008; Di Cera, 2009 for reviews). A growing number of studies demonstrate that these protein systems are widely expressed in many cell types and regions in mammalian brains. Accumulating lines of evidence suggest that the brain has co-opted the activities of these interesting proteins to regulate various processes underlying synaptic activity and behavior. In this review, we discuss emerging roles for serine proteases in the regulation of mechanisms underlying synaptic plasticity and memory formation.

  5. Comparative analysis on the distribution of protease activities among fruits and vegetable resources.

    Science.gov (United States)

    Sun, Qian; Zhang, Bin; Yan, Qiao-Juan; Jiang, Zheng-Qiang

    2016-12-15

    In this study, a comparative analysis on the distribution of protease activities among 90 plant resources, including fruits and vegetables, has been performed. Protease activities of plant extracts were assayed at different pH values (pH 3.0, pH 7.5 and pH 10.5) using casein as a substrate. Ten fruits and thirteen vegetables show protease activities above 10U/g. Pineapple, fig and papaya, which are used for commercial protease production, exhibited high protease activities. Additionally, high protease activities were detected in kiwifruit (28.8U/g), broccoli (16.9U/g), ginger (16.6U/g), leek (32.7U/g) and red pepper (15.8U/g) at different pH values. SDS-PAGE and zymograms confirmed that various types of proteases existed in the five plant extracts and might be explored. Furthermore, five plant extracts were treated by different protease inhibitors. These results show that there are still many plant resources unexplored, which may be promising candidates for plant-derived protease production.

  6. Post-prandial protease activity in the digestive tract of African catfish, Clarias gariepinus, larvae fed decapsulated cysts of Artemia

    NARCIS (Netherlands)

    García Ortega, A.; Verreth, J.; Segner, H.

    2000-01-01

    The alkaline proteolytic activity in the gut of African catfish larvae was studied during short time ranges from 30 min to 4 h after ingestion of decapsulated Artemia cysts. The variation in total protease and trypsin activities during the day was monitored during starvation, after one single meal i

  7. MALT1 Protease Activity Is Required for Innate and Adaptive Immune Responses.

    Directory of Open Access Journals (Sweden)

    Jong W Yu

    Full Text Available CARMA-BCL10-MALT1 signalosomes play important roles in antigen receptor signaling and other pathways. Previous studies have suggested that as part of this complex, MALT1 functions as both a scaffolding protein to activate NF-κB through recruitment of ubiquitin ligases, and as a protease to cleave and inactivate downstream inhibitory signaling proteins. However, our understanding of the relative importance of these two distinct MALT1 activities has been hampered by a lack of selective MALT1 protease inhibitors with suitable pharmacologic properties. To fully investigate the role of MALT1 protease activity, we generated mice homozygous for a protease-dead mutation in MALT1. We found that some, but not all, MALT1 functions in immune cells were dependent upon its protease activity. Protease-dead mice had defects in the generation of splenic marginal zone and peritoneal B1 B cells. CD4+ and CD8+ T cells displayed decreased T cell receptor-stimulated proliferation and IL-2 production while B cell receptor-stimulated proliferation was partially dependent on protease activity. In dendritic cells, stimulation of cytokine production through the Dectin-1, Dectin-2, and Mincle C-type lectin receptors was also found to be partially dependent upon protease activity. In vivo, protease-dead mice had reduced basal immunoglobulin levels, and showed defective responses to immunization with T-dependent and T-independent antigens. Surprisingly, despite these decreased responses, MALT1 protease-dead mice, but not MALT1 null mice, developed mixed inflammatory cell infiltrates in multiple organs, suggesting MALT1 protease activity plays a role in immune homeostasis. These findings highlight the importance of MALT1 protease activity in multiple immune cell types, and in integrating immune responses in vivo.

  8. MALT1 Protease Activity Is Required for Innate and Adaptive Immune Responses.

    Science.gov (United States)

    Yu, Jong W; Hoffman, Sandy; Beal, Allison M; Dykon, Angela; Ringenberg, Michael A; Hughes, Anna C; Dare, Lauren; Anderson, Amber D; Finger, Joshua; Kasparcova, Viera; Rickard, David; Berger, Scott B; Ramanjulu, Joshi; Emery, John G; Gough, Peter J; Bertin, John; Foley, Kevin P

    2015-01-01

    CARMA-BCL10-MALT1 signalosomes play important roles in antigen receptor signaling and other pathways. Previous studies have suggested that as part of this complex, MALT1 functions as both a scaffolding protein to activate NF-κB through recruitment of ubiquitin ligases, and as a protease to cleave and inactivate downstream inhibitory signaling proteins. However, our understanding of the relative importance of these two distinct MALT1 activities has been hampered by a lack of selective MALT1 protease inhibitors with suitable pharmacologic properties. To fully investigate the role of MALT1 protease activity, we generated mice homozygous for a protease-dead mutation in MALT1. We found that some, but not all, MALT1 functions in immune cells were dependent upon its protease activity. Protease-dead mice had defects in the generation of splenic marginal zone and peritoneal B1 B cells. CD4+ and CD8+ T cells displayed decreased T cell receptor-stimulated proliferation and IL-2 production while B cell receptor-stimulated proliferation was partially dependent on protease activity. In dendritic cells, stimulation of cytokine production through the Dectin-1, Dectin-2, and Mincle C-type lectin receptors was also found to be partially dependent upon protease activity. In vivo, protease-dead mice had reduced basal immunoglobulin levels, and showed defective responses to immunization with T-dependent and T-independent antigens. Surprisingly, despite these decreased responses, MALT1 protease-dead mice, but not MALT1 null mice, developed mixed inflammatory cell infiltrates in multiple organs, suggesting MALT1 protease activity plays a role in immune homeostasis. These findings highlight the importance of MALT1 protease activity in multiple immune cell types, and in integrating immune responses in vivo.

  9. MALT1 Protease Activity Is Required for Innate and Adaptive Immune Responses

    Science.gov (United States)

    Yu, Jong W.; Hoffman, Sandy; Beal, Allison M.; Dykon, Angela; Ringenberg, Michael A.; Hughes, Anna C.; Dare, Lauren; Anderson, Amber D.; Finger, Joshua; Kasparcova, Viera; Rickard, David; Berger, Scott B.; Ramanjulu, Joshi; Emery, John G.; Gough, Peter J.; Bertin, John; Foley, Kevin P.

    2015-01-01

    CARMA-BCL10-MALT1 signalosomes play important roles in antigen receptor signaling and other pathways. Previous studies have suggested that as part of this complex, MALT1 functions as both a scaffolding protein to activate NF-κB through recruitment of ubiquitin ligases, and as a protease to cleave and inactivate downstream inhibitory signaling proteins. However, our understanding of the relative importance of these two distinct MALT1 activities has been hampered by a lack of selective MALT1 protease inhibitors with suitable pharmacologic properties. To fully investigate the role of MALT1 protease activity, we generated mice homozygous for a protease-dead mutation in MALT1. We found that some, but not all, MALT1 functions in immune cells were dependent upon its protease activity. Protease-dead mice had defects in the generation of splenic marginal zone and peritoneal B1 B cells. CD4+ and CD8+ T cells displayed decreased T cell receptor-stimulated proliferation and IL-2 production while B cell receptor-stimulated proliferation was partially dependent on protease activity. In dendritic cells, stimulation of cytokine production through the Dectin-1, Dectin-2, and Mincle C-type lectin receptors was also found to be partially dependent upon protease activity. In vivo, protease-dead mice had reduced basal immunoglobulin levels, and showed defective responses to immunization with T-dependent and T-independent antigens. Surprisingly, despite these decreased responses, MALT1 protease-dead mice, but not MALT1 null mice, developed mixed inflammatory cell infiltrates in multiple organs, suggesting MALT1 protease activity plays a role in immune homeostasis. These findings highlight the importance of MALT1 protease activity in multiple immune cell types, and in integrating immune responses in vivo. PMID:25965667

  10. Feces derived allergens of Tyrophagus putrescentiae reared on dried dog food and evidence of the strong nutritional interaction between the mite and Bacillus cereus producing protease bacillolysins and exo-chitinases

    Directory of Open Access Journals (Sweden)

    Tomas eErban

    2016-02-01

    Full Text Available Tyrophagus putrescentiae (Schrank, 1781 is an emerging source of allergens in stored products and homes. Feces proteases are the major allergens of astigmatid mites (Acari: Acaridida. In addition, the mites are carriers of microorganisms and microbial adjuvant compounds that stimulate innate signaling pathways. We sought to analyze the mite feces proteome, proteolytic activities and mite-bacterial interaction in dry dog food. Proteomic methods comprising enzymatic and zymographic analysis of proteases and 2D-E-MS/MS were performed. The highest protease activity was assigned to trypsin-like proteases; lower activity was assigned to chymotrypsin-like proteases, and the cysteine protease cathepsin B-like had very low activity. The 2D-E-MS/MS proteomic analysis identified mite trypsin allergen Tyr p3, fatty acid-binding protein Tyr p13 and putative mite allergens ferritin (Grp 30 and (polyubiquitins. Tyr p3 was detected at different positions of the 2D-E. It indicates presence of zymogen at basic pI, and mature-enzyme form and enzyme fragment at acidic pI. Bacillolysins (neutral and alkaline proteases of Bacillus cereus symbiont can contribute to the protease activity of the mite extract. The bacterial exo-chitinases likely contribute to degradation of mite exuviae, mite bodies or food boluses consisting of chitin, including the peritrophic membrane. Thus, the chitinases disrupt the feces and facilitate release of the allergens. B. cereus was isolated and identified based on amplification and sequencing of 16S rRNA and motB genes. B. cereus was added into high-fat, high-protein (dry dog food and low-fat, low-protein (flour diets to 1% and 5% (w/w, and the diets palatability was evaluated in 21-day population growth test. The supplementation of diet with B. cereus significantly suppressed population growth and the suppressive effect was higher in the high-fat, high-protein diet than in the low-fat, low-protein food. Thus, B. cereus has to coexist

  11. Feces Derived Allergens of Tyrophagus putrescentiae Reared on Dried Dog Food and Evidence of the Strong Nutritional Interaction between the Mite and Bacillus cereus Producing Protease Bacillolysins and Exo-chitinases.

    Science.gov (United States)

    Erban, Tomas; Rybanska, Dagmar; Harant, Karel; Hortova, Bronislava; Hubert, Jan

    2016-01-01

    Tyrophagus putrescentiae (Schrank, 1781) is an emerging source of allergens in stored products and homes. Feces proteases are the major allergens of astigmatid mites (Acari: Acaridida). In addition, the mites are carriers of microorganisms and microbial adjuvant compounds that stimulate innate signaling pathways. We sought to analyze the mite feces proteome, proteolytic activities, and mite-bacterial interaction in dry dog food (DDF). Proteomic methods comprising enzymatic and zymographic analysis of proteases and 2D-E-MS/MS were performed. The highest protease activity was assigned to trypsin-like proteases; lower activity was assigned to chymotrypsin-like proteases, and the cysteine protease cathepsin B-like had very low activity. The 2D-E-MS/MS proteomic analysis identified mite trypsin allergen Tyr p3, fatty acid-binding protein Tyr p13 and putative mite allergens ferritin (Grp 30) and (poly)ubiquitins. Tyr p3 was detected at different positions of the 2D-E. It indicates presence of zymogen at basic pI, and mature-enzyme form and enzyme fragment at acidic pI. Bacillolysins (neutral and alkaline proteases) of Bacillus cereus symbiont can contribute to the protease activity of the mite extract. The bacterial exo-chitinases likely contribute to degradation of mite exuviae, mite bodies or food boluses consisting of chitin, including the peritrophic membrane. Thus, the chitinases disrupt the feces and facilitate release of the allergens. B. cereus was isolated and identified based on amplification and sequencing of 16S rRNA and motB genes. B. cereus was added into high-fat, high-protein (DDF) and low-fat, low-protein (flour) diets to 1 and 5% (w/w), and the diets palatability was evaluated in 21-day population growth test. The supplementation of diet with B. cereus significantly suppressed population growth and the suppressive effect was higher in the high-fat, high-protein diet than in the low-fat, low-protein food. Thus, B. cereus has to coexist with the mite in

  12. 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.

  13. Development and validation of stability-indicating RP-HPLC method for determination of Olmesartan medoxomile in pharmaceutical dosage form and identification, characterization of alkaline degradation impurity of Olmesartan medoxomile drug substance as well as drug product

    Directory of Open Access Journals (Sweden)

    Jain P.S.

    2012-01-01

    Full Text Available Olmesartan Medoxomil (OLME belongs to a group of angiotensin II receptor blockers used as an antihypertensive agent and is currently being used for prevention of Hypertension. This paper describes the Validation and development of stability indicating RP-HPLC method for the determination of OLME in the presence of its degradation products generated from forced degradation study and characterization of degradation product (impurity. The assay involved gradient elution of OLME on An LC GC BDS C18 column (250 × 4.5mm, 5-μm particle size was employed for loading the sample. The mobile phase A consists of 7 ml Triethylamine in 1000 ml water (pH adjusted to 3.0 with orthophosphoric acid and B contains acetonitrile. Quantification was achieved with photodiode array detection at 257 nm. The chromatographic separation was obtained with a retention time of 6.72 min, and the method was linear in the range 50-150 µg/ml. The method was validated according to the ICH guidelines with respect to linearity, precision, accuracy, limit of detection (LOD, limit of quantification (LOQ, specificity and robustness. Impurity found in stressed and stability studies of Olmesartan Medoxomil in both drug substance and drug product are described. This degradation product is identified as 1-(biphenyl-4-ylmethyl-1H-imidazole-5-carboxylic acid. An Alkaline degradation pathway of Olmesartan medoxomil, for the formation of this degradation product, has been proposed and degradation product was characterized.

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

    Science.gov (United States)

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

    2016-04-01

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

  15. 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

  16. Statistical and evolutionary optimization for enhanced production of an antileukemic enzyme, L-asparaginase, in a protease-deficient Bacillus aryabhattai ITBHU02 isolated from the soil contaminated with hospital waste.

    Science.gov (United States)

    Singh, Yogendra; Srivastav, S K

    2013-04-01

    Over the past few decades, L-asparaginase has emerged as an excellent anti-neoplastic agent. In present study, a new strain ITBHU02, isolated from soil site near degrading hospital waste, was investigated for the production of extracellular L-asparaginase. Further, it was renamed as Bacillus aryabhattai ITBHU02 based on its phenotypical features, biochemical characteristics, fatty acid methyl ester (FAME) profile and phylogenetic similarity of 16S rDNA sequences. The strain was found protease-deficient and its optimal growth occurred at 37 degrees C and pH 7.5. The strain was capable of producing enzyme L-asparaginase with maximum specific activity of 3.02 +/- 0.3 Umg(-1) protein, when grown in un-optimized medium composition and physical parameters. In order to improve the production of L-asparaginase by the isolate, response surface methodology (RSM) and genetic algorithm (GA) based techniques were implemented. The data achieved through the statistical design matrix were used for regression analysis and analysis of variance studies. Furthermore, GA was implemented utilizing polynomial regression equation as a fitness function. Maximum average L-asparaginase productivity of 6.35 Umg(-1) was found at GA optimized concentrations of 4.07, 0.82, 4.91, and 5.2 gL(-1) for KH2PO4, MgSO4 x 7H2O, L-asparagine, and glucose respectively. The GA optimized yield of the enzyme was 7.8% higher in comparison to the yield obtained through RSM based optimization.

  17. Stable earthworm serine proteases: application of the protease function and usefulness of the earthworm autolysate.

    Science.gov (United States)

    Nakajima, N; Sugimoto, M; Ishihara, K

    2000-01-01

    The fibrinolytic enzymes from Lumbricus rubellus [Nakajima, N. et al., Biosci. Biotechnol. Biochem., 57, 1726-1730 (1993), 60, 293-300 (1996), and 63, 2031-2033 (1999)] were further characterized to exploit their catalytic functions. These enzymes are stable in solution for long periods at room temperature and strongly resistant to organic solvents, even toluene and n-hexane. The serine proteases can act on various protein substrates such as elastin and hemoglobin as well as fibrin, and also catalyzed the hydrolysis of esters such as ethyl acetate and a bioplastic, poly[(R)-3-hydroxybutyrate] film. The enzymes, in the absence of microbial degradation, contributed to the production of the earthworm autolysate possessing antioxidant ability and protease activity, whose components were similar to those of soy sauce. The extract of the earthworm autolysate could be used as a peptone substitute in media for the cultivation of microorganisms.

  18. A Comparison between the Effects of Albendazole and Meben¬ dazole on the Enzymatic Activity of Excretory / Secretory Prod-ucts of Echinococcus granulosus Protoscoleces in Vitro

    Directory of Open Access Journals (Sweden)

    Seyed Jafar ADNANI SADATI

    2016-02-01

    Full Text Available Background: Hydatid cysts are formed in human body can be treated clinically by surgery or drugs such as albendazole (ABZ and mebendazole (MBZ. The purpose of this study was comparing the effects of ABZ and MBZ on glutathione-S-transferase, alkaline phosphatase and protease enzymes activities in protoscoleces of hydatid cyst. Methods: The culture supernatants containing the parasite Excretory / Secretory (E/S products were collected every 12 h for 72 h. The E/S products of treated samples with 1µg/ml ABZ and MBZ and the control one were collected and after centrifugation then protein concentrations were measured according to Bradford method. GST, ALP and protease activities of E/S products were assessed photometrically.Results: The mean of GST specific activity level in treated protoscoleces with ABZ and MBZ and in control group were obtained 69.44, 132.83 and 225.47U/mg/protein/ml respectively. The mean ALP activity level in treated protoscoleces with ABZ and MBZ and in control group were detected 19.22, 22.27 and 27.85 U/mg/protein/ml respectively. The protease activity level in treated protoscoleces with ABZ and MBZ were not detected. While the mean of protease activity level in control group was 7.61U/mg/proteins. Statistical analysis showed the significant difference between protein concentrations, the specific activities of GST, ALP and protease enzymes in treated protoscoleces in comparison with control group (P<0.05. Also, the significant difference were seen between specific activities of GST and ALP enzymes in treated protoscoleces with ABZ in comparison with treated group with MBZ (P<0.05.Conclusion: ABZ is more effective on the enzymes activities (GST and ALP as compared with MBZ. Keywords: Hydatid cyst protoscoleces, Albendazole, Mebandazole, Protease, Glutathione S-Transferase, Alkaline phosphatase

  19. Research Progress on Natural Products as Corrosion Inhibitor in Acid, Neutral and Alkaline Mediums%酸性、中性、碱性介质中植物型缓蚀剂的研究进展

    Institute of Scientific and Technical Information of China (English)

    郑杰; 王亚娜; 章柏林; 杨婷; 焦学敏; 何勇

    2011-01-01

    Plant extracts have become important as an environmentally acceptable, readily available and renewable source for wide range of inhibitors. In general, the plant extracts are of inhibitors with high inhibition efficiency and of non toxicant. This article summarizes the reaction mechanism and gives a vivid account of natural products which are used as corrosion inhibitors in acid, neutral and alkaline mediums. The prospects of the development trend are also discussed.%植物型缓蚀剂具有成本低,来源广,无毒,高效等特点,是未来缓蚀剂的发展方向.本文概括介绍了植物型缓蚀剂的作用机理,总结了植物型缓蚀剂在酸性、中性和碱性介质中的研究进展,并对今后的研究提出了展望.

  20. Reaction mechanism of -acylhydroxamate with cysteine proteases

    Indian Academy of Sciences (India)

    R Shankar; P Kolandaivel

    2007-09-01

    The gas-phase reaction mechanism of -acylhydroxamate with cysteine proteases has been investigated using ab initio and density functional theory. On the irreversible process, after breakdown of tetrahedral intermediate (INT1), small 1-2 anionotropic has been formed and rearranged to give stable by-products sulfenamide (P1) and thiocarbamate (P2) with considerable energy loss. While, on the reversible part of this reaction mechanism, intermediate (INT2) breaks down on oxidation, to form a stable product (P3). Topological and AIM analyses have been performed for hydrogen bonded complex in this reaction profile. Intrinsic reaction coordinates [IRC, minimum-energy path (MEP)] calculation connects the transition state between R-INT1, INT1-P1 and INT1-P2. The products P1, P2 and P3 are energetically more stable than the reactant and hence the reaction enthalpy is found to be exothermic.

  1. 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

  2. [Cytokines and proteases involved in pathogenesis of COPD].

    Science.gov (United States)

    Yamaya, Atsuyo; Osanai, Kazuhiro

    2011-10-01

    COPD is characterized by persistence of chronic inflammation in small airways and alveoli. Macrophages, neutrophils, and a specialized subset of T lymphocytes orchestrate the mild inflammation. This article focuses on humoral factors such as cytokines and chemokines that recruit these inflammatory and immune cells to the lungs, and proteases/antiproteases that ultimately cause structural derangement in the terminal respiratory zones. In addition to the classical protease and antiprotease imbalance hypothesis, alveolar homeostasis abnormality that comes from imbalance of lung constitutional cell apoptosis and regeneration may play a role in emphysema development. Also, autoimmunity to elastin degradation products may take part in the disease.

  3. Protease Inhibitors Do Not Affect Antibody Responses to Pneumococcal Vaccination.

    Science.gov (United States)

    De La Rosa, Indhira; Munjal, Iona M; Rodriguez-Barradas, Maria; Yu, Xiaoying; Pirofski, Liise-Anne; Mendoza, Daniel

    2016-06-01

    HIV(+) subjects on optimal antiretroviral therapy have persistently impaired antibody responses to pneumococcal vaccination. We explored the possibility that this effect may be due to HIV protease inhibitors (PIs). We found that in humans and mice, PIs do not affect antibody production in response to pneumococcal vaccination.

  4. Space-time variability of alkalinity in the Mediterranean Sea

    Directory of Open Access Journals (Sweden)

    G. Cossarini

    2014-09-01

    Full Text Available The paper provides a basin assessment of the spatial distribution of ocean alkalinity in the Mediterranean Sea. The assessment is made using a 3-D transport-biogeochemical-carbonate model to integrate the available experimental findings, which also constrains model output. The results indicate that the Mediterranean Sea shows alkalinity values that are much higher than those observed in the Atlantic Ocean on a basin-wide scale. A marked west-to-east surface gradient of alkalinity is reproduced as a response to the terrestrial discharges, the mixing effect with the Atlantic water entering from the Gibraltar Strait and the Black Sea water from Dardanelles, and the surface flux of evaporation minus precipitation. Dense water production in marginal seas (Adriatic and Aegean Seas, where alkaline inputs are relevant, and the Mediterranean thermohaline circulation sustains the west-to-east gradient along the entire water column. In the surface layers, alkalinity has a relevant seasonal cycle (up to 40 μmol kg−1 that is driven both by physical and biological processes. A comparison of alkalinity vs. salinity indicates that different regions present different relationships. In regions of freshwater influence, the two measures are negatively correlated due to riverine alkalinity input, whereas they are positively correlated in open seas. Alkalinity always is much higher than in the Atlantic waters, which might indicate a higher than usual buffering capacity towards ocean acidification, even at high concentrations of dissolved inorganic carbon.

  5. 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.

  6. Peptide synthesis in neat organic solvents with novel thermostable proteases.

    Science.gov (United States)

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

    2015-06-01

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

  7. Allostery in trypsin-like proteases suggests new therapeutic strategies.

    Science.gov (United States)

    Gohara, David W; Di Cera, Enrico

    2011-11-01

    Trypsin-like proteases (TLPs) are a large family of enzymes responsible for digestion, blood coagulation, fibrinolysis, development, fertilization, apoptosis and immunity. A current paradigm posits that the irreversible transition from an inactive zymogen to the active protease form enables productive interaction with substrate and catalysis. Analysis of the entire structural database reveals two distinct conformations of the active site: one fully accessible to substrate (E) and the other occluded by the collapse of a specific segment (E*). The allosteric E*-E equilibrium provides a reversible mechanism for activity and regulation in addition to the irreversible zymogen to protease conversion and points to new therapeutic strategies aimed at inhibiting or activating the enzyme. In this review, we discuss relevant examples, with emphasis on the rational engineering of anticoagulant thrombin mutants.

  8. A practical total synthesis of the microbial alkaline proteinase inhibitor (MAPI).

    Science.gov (United States)

    Haebich, Dieter; Hillisch, Alexander; El Sheikh, Sherif

    2009-12-01

    Diverse serine and cysteine proteases as well as alkaline proteinases and elastases play a crucial role in numerous biological processes. Natural peptide aldehydes such as the "microbial alkaline proteinase inhibitor" (MAPI, 1) are valuable tools to characterize novel enzymes and to study their function in nature. Within a drug discovery program we wanted to design and explore non-natural MAPI congeners with novel biological profiles. To that end we devised a simple, practical, and scalable synthesis of MAPI 1 from readily available amino acid building blocks. The modular nature of our approach allows convenient structural modification of the MAPI backbone.

  9. 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.

  10. An alkaline element

    Energy Technology Data Exchange (ETDEWEB)

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

    1983-04-28

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

  11. 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…

  12. 极地适冷菌Pseudoalteromonas sp.QI-1产适冷蛋白酶发酵条件的优化%Optimization of cold-active protease production by polar psychrotrophic bacterium Pseudoalteromonas sp.QI-1

    Institute of Scientific and Technical Information of China (English)

    徐国英; 林学政; 王能飞; 朱启忠

    2011-01-01

    The culture conditions of polar psychrotrophic bacterium Pseudoalteromonas sp. QI-1 were optimized for the production of cold-active protease. The optimum fermentation conditions were determined as follows: optimal temperatures for growth and enzyme production were both at 5 ℃, inoculation volume was 1% ,and liquid volume was 10% at pH 5. Culture medium for protease production of strain QI-1 was also studied. Results showed that nitrogen source and carbon source preferred by protease production were bran and sodium acetate. 2% NaC1,10 mmol/L Mg2+ ,0.5% Tween-80 and 0.75% casein were suitable for the enzyme production. The orthogonal experiment design showed that the optimum medium (per liter) consisted of 5 g bran,2.5 g yeast extract,3 g casein,3 g MgCl2 ·6H20 and 1.5 g KCl. Under the optimum condition, the protease activity was up to 166. 20 U/mL and the yield of protease increased about 56%.%对极地适冷菌Pseudoalteromonas sp.QI-1产适冷蛋白酶的发酵条件进行优化.结果表明:菌株QI-1的最适生长和产酶温度均为5℃;最佳接种量为1%;发酵培养基的最适初始pH和最佳装样量分别为5和10%;盐度为2%时对菌株的生长和产酶最为有利;麸皮和醋酸钠分别为最佳N源和C源;添加0.75%酪蛋白时菌株QI-1胞外蛋白酶的活性最高;10 mmol/L Mg2+和0.5%Tween-80有利于产酶.正交试验结果表明:菌株Pseudoalteromonas sp.QI-1产蛋白酶较佳培养基配方(g/L)为麸皮5,酵母粉2.5,酪蛋白3,MgCl2·6H2O3,KCl 1.5;发酵液比酶活为166.20 U/mL,较优化前提高了约56%.

  13. THE USE OF DIFFERENT PROTEASES TO HYDROLYZE GLIADINS

    Directory of Open Access Journals (Sweden)

    Peter Socha

    2015-02-01

    Full Text Available Gliadins represent alcohol-soluble fraction of wheat storage proteins which is responsible for development of celiac disease. The only and effective treatment for celiac disease is strict adherence to a gluten-free diet excluding any food made with wheat, as well as rye, barley and possibly oat flour. Enzymatic modification of wheat gliadins seems to be an alternative method for decreasing of celiac activity. The aim of our study was a trial of enzymatic modification of wheat gliadins using fungal (Aspergillus sp., Aspergillus oryzae, Aspergillus niger and bacterial (Bacillus licheniformis, Bacillus stearothermophilus, Bacillus thermoproteolyticus, Streptomyces griseus proteases. The reaction was performed up to 60 min, stopped by addition of appropriate synthetic inhibitor and products of limited proteolysis were analyzed by SDS-PAGE method. From fungal proteases most effective proteolytic activity was observed using acid proteinase from A. niger since wheat gliadins and low molecular weight peptides were completely degraded. Bacterial proteases form B. licheniformis and B. thermoproteolyticus acted very effective and as the result of hydrolysis, the products of lower molecular weight (<15 kDa occurred. Most of the wheat gliadins were susceptible to proteolysis by examined bacterial enzymes (exception were protease from B. stearothermophilus and S. griseus. Although wheat gliadins are susceptible to enzymatic degradation, further analysis (e.g. immunochemical or mass spectrometry are desirable to confirm if the products of proteolysis have lost or at least partially decrease their celiac activity.

  14. 蛋白酶的研究进展%The Research Progress of Protease

    Institute of Scientific and Technical Information of China (English)

    巩晓芳; 张宗舟; 薛林贵

    2011-01-01

    蛋白酶在工业生产和科学研究以及人们的日常生活中都扮演着重要的角色,起着非常重要的作用。目前许多科学研究者都致力于蛋白酶的研究,包括蛋白酶的生产研究和蛋白酶的应用研究,以期提高蛋白酶的产量和拓宽蛋白酶的应用领域来解决更多的与蛋白质相关的生产问题和资源供给问题。文章从蛋白酶的理化性质,蛋白酶的来源及种类,蛋白酶固定化技术的研究以及蛋白酶的应用进行了综述,最后对蛋白酶的研究进行了展望。%Protease has played an important role and made a good function in industrial production and scientific research as well as in people's daily life. Now many of the scientific researchers are working in protease study, including production and applications about protease, to enhance the output of protease and expand the application field of protease to solve more problems about production that related protein material and the resources supply in social. This article reviewed the properties of protease from physical to chemical, the source and species of protease, the immobilized technology research about protease and the application of protease, then presented the research of protease.

  15. Response of Desulfovibrio vulgaris to Alkaline Stress

    Energy Technology Data Exchange (ETDEWEB)

    Stolyar, S.; He, Q.; He, Z.; Yang, Z.; Borglin, S.E.; Joyner, D.; Huang, K.; Alm, E.; Hazen, T.C.; Zhou, J.; Wall, J.D.; Arkin, A.P.; Stahl, D.A.

    2007-11-30

    The response of exponentially growing Desulfovibrio vulgarisHildenborough to pH 10 stress was studied using oligonucleotidemicroarrays and a study set of mutants with genes suggested by microarraydata to be involved in the alkaline stress response deleted. The datashowed that the response of D. vulgaris to increased pH is generallysimilar to that of Escherichia coli but is apparently controlled byunique regulatory circuits since the alternative sigma factors (sigma Sand sigma E) contributing to this stress response in E. coli appear to beabsent in D. vulgaris. Genes previously reported to be up-regulated in E.coli were up-regulated in D. vulgaris; these genes included three ATPasegenes and a tryptophan synthase gene. Transcription of chaperone andprotease genes (encoding ATP-dependent Clp and La proteases and DnaK) wasalso elevated in D. vulgaris. As in E. coli, genes involved in flagellumsynthesis were down-regulated. The transcriptional data also identifiedregulators, distinct from sigma S and sigma E, that are likely part of aD. vulgaris Hildenborough-specific stress response system.Characterization of a study set of mutants with genes implicated inalkaline stress response deleted confirmed that there was protectiveinvolvement of the sodium/proton antiporter NhaC-2, tryptophanase A, andtwo putative regulators/histidine kinases (DVU0331 andDVU2580).

  16. Role of Allergen Source-Derived Proteases in Sensitization via Airway Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Yasuhiro Matsumura

    2012-01-01

    Full Text Available Protease activity is a characteristic common to many allergens. Allergen source-derived proteases interact with lung epithelial cells, which are now thought to play vital roles in both innate and adaptive immune responses. Allergen source-derived proteases act on airway epithelial cells to induce disruption of the tight junctions between epithelial cells, activation of protease-activated receptor-2, and the production of thymic stromal lymphopoietin. These facilitate allergen delivery across epithelial layers and enhance allergenicity or directly activate the immune system through a nonallergic mechanism. Furthermore, they cleave regulatory cell surface molecules involved in allergic reactions. Thus, allergen source-derived proteases are a potentially critical factor in the development of allergic sensitization and appear to be strongly associated with heightened allergenicity.

  17. Characterization of two cysteine proteases secreted by Blastocystis ST7, a human intestinal parasite.

    Science.gov (United States)

    Wawrzyniak, Ivan; Texier, Catherine; Poirier, Philippe; Viscogliosi, Eric; Tan, Kevin S W; Delbac, Frédéric; El Alaoui, Hicham

    2012-09-01

    Blastocystis spp. are unicellular anaerobic intestinal parasites of both humans and animals and the most prevalent ones found in human stool samples. Their association with various gastrointestinal disorders raises the questions of its pathogenicity and of the molecular mechanisms involved. Since secreted proteases are well-known to be implicated in intestinal parasite virulence, we intended to determine whether Blastocystis spp. possess such pathogenic factors. In silico analysis of the Blastocystis subtype 7 (ST7) genome sequence highlighted 22 genes coding proteases which were predicted to be secreted. We characterized the proteolytic activities in the secretory products of Blastocystis ST7 using specific protease inhibitors. Two cysteine proteases, a cathepsin B and a legumain, were identified in the parasite culture supernatant by gelatin zymographic SDS-PAGE gel and MS/MS analysis. These proteases might act on intestinal cells and disturb gut function. This work provides serious molecular candidates to link Blastocystis spp. and intestinal disorders.

  18. Impact of protease activity of yeasts on wine fermentation and formation of volatile and non-volatile metabolites

    OpenAIRE

    Chomsri, Ni-orn

    2008-01-01

    Fifty yeast strains previously isolated from grapes and wines were investigated for extracellular protease activity. Saccharomyces yeasts showed no detectable protease activity, while most of the non-Saccharomyces yeasts exhibited proteolytic activity. Nine of the fifty yeast strains were cultivated in grape juice and were analysed for the production of extracellular proteases. The proteolytic activity of Metschnikowia pulcherrima (M 004) was as high as that of Rhodotolura sp. (R3). Very low ...

  19. 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.

  20. Isolation and partial characterization of a protease from Agave americana variegata.

    Science.gov (United States)

    Du Toit, P J

    1976-05-13

    A new protease was isolated from an extract of leaves of Agave americana variegata. The protease (EC 3.4.-) was purified 565-fold with a yield of 39.5%. The 43.8 mg enzyme had a specific activity of 0.44 units/mg. According to electrophoretic, ultracentrifugal and other physical characterizations the enzyme was homogeneous. The enzyme had a MR of 57000, a S20,W-value of 4.37 S, a D20, W-value of 6.8-7.0 - 10(-7) cm2sec-1, a Stokes radius of 3.18 nm, a partial specific volume of 0.735 cm3g-1, a frictional ration of 1.25, a molecular absorbancy index at 280 nm of 5.773-10(4), an isoelectric point of 5.25 and contained 8-10% carbohydrate. The enzyme contained no cysteine. Agave protease could hydrolyze a variety of protein substrates although it did have a restricted specificity. It is not a sulphhydryl protease but seems to be an alkaline "serine" protease with an optimum pH of 7.8-8.0 Agave protease had marked esterolytic activity and with Cbz-Tyr-ONp had an apparent Michaelis constant of 0.0345 -10(-3) M and a V of 1.24 mol substrate/mol enzyme per sec. The enzyme did not need metal ions for optimal activity, monovalent cations did not influence its kinetic parameters, but it was inhibited by cobalt, pC1HgBzO- and TosPheCH2C1. With respect to its primary specificity, as well as its pH-dependence there was a resemblance with chymotrypsin, although the rate of hydrolysis of Agave protease is much lower.

  1. 复合蛋白酶在卤鹅类潮式卤水生产中的应用%Application of Protease Complex in the Production of Chaozhou Brine Goose

    Institute of Scientific and Technical Information of China (English)

    陈宇; 郭卓钊; 郭美媛; 郭奕纯; 杨曼; 黄妙云

    2012-01-01

    针对蛋白酶在潮式卤鹅传统熟肉食品生产中的应用,以剪切力的变化来研究木瓜蛋白酶、胰蛋白酶和复合蛋白酶对卤鹅肉质的影响.通过单因素试验和正交试验确定了最佳的工艺生产条件为:复合酶液的胰蛋白酶用量为2000 U/kg原料、木瓜蛋白酶用量为1000 U/kg原料,温度为20℃,处理时间为30 min,所得到的卤鹅颜色酱红有光泽、肉质细嫩有弹性,滋味浓郁.%This paper described the application of protease complex in the traditional Chaozhou brine goose, by analysis of the change of shearing force after dealing by papain, trypsin and mixed proteases. The optional conditions were obtained by orthogonal experiments as follows: trypsin in mixed proteases 2000 U/kg, papain in mixed proteases 1000 U/kg, temperature 20 ℃and time 30 min. The brine goose had a glossy garnet red color, and the meat showed a delicious and elastic taste with rich flavor.

  2. EFFECTIVE ALKALINE PEROXIDE OXIDATION PRETREATMENT OF SHEA TREE SAWDUST FOR THE PRODUCTION OF BIOFUELS: KINETICS OF DELIGNIFICATION AND ENZYMATIC CONVERSION TO SUGAR AND SUBSEQUENT PRODUCTION OF ETHANOL BY FERMENTATION USING Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    A. O. Ayeni

    Full Text Available Abstract Shea tree sawdust delignification kinetic data during alkaline peroxide pretreatment were investigated at temperatures of 120 °C, 135 °C, and 150 °C. The activation energy during delignification was 76.4 kJ/mol and the Arrhenius constant was calculated as 8.4 x 106 per min. The reducing sugar yield for the treated to the untreated biomass was about 22-fold. Enzymatic hydrolysis conditions studied were; time (72 h and 96 h, substrate concentration (20, 30, 40, and 50 g/L, and enzyme loadings (10, 25, 40, 50 FPU/g dry biomass, which showed the optimum conditions of 96 h, 40 g/L, and 25 FPU/g dry biomass at 45 °C hydrolysis temperature. At the optimized enzymatic hydrolysis conditions, the reducing sugar yield was 416.32 mg equivalent glucose/g treated dry biomass. After 96 h fermentation of treated biomass, the ethanol obtained at 2% effective cellulose loading was 12.73 g/L. Alkaline peroxide oxidation pretreatment and subsequent enzymatic hydrolysis improved the ethanol yield of the biomass.

  3. Inhibitors of lysosomal cysteine proteases

    Directory of Open Access Journals (Sweden)

    Lyanna O. L.

    2011-04-01

    Full Text Available The review is devoted to the inhibitors of cysteine proteinases which are believed to be very important in many biochemical processes of living organisms. They participate in the development and progression of numerous diseases that involve abnormal protein turnover. One of the main regulators of these proteinases is their specific inhibitors: cystatins. The aim of this review was to present current knowledge about endogenous inhibitors of lysosomal cysteine proteases and their synthetic analogs.

  4. Nelfinavir: fourth protease inhibitor approved.

    Science.gov (United States)

    1997-01-01

    The Food and Drug Administration (FDA) has granted accelerated approval to nelfinavir in both adult and pediatric formulations. Agouron, the manufacturer, used innovative computerized drug design techniques to discover, design, and refine the nelfinavir molecule. Nelfinavir is marketed under the trade name Viracept, and costs $5,000 per year. Early clinical trials find it to be as powerful as the other protease inhibitors, but with a different resistance profile. The drug has relatively few drug indications; however, several compounds have been contraindicated.

  5. 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.

  6. Production and partial characterization of alkaline polygalacturonase secreted by thermophilic Bacillus sp. SMIA-2 under submerged culture using pectin and corn steep liquor

    Directory of Open Access Journals (Sweden)

    Marcela Vicente Vieira de Andrade

    2011-03-01

    Full Text Available Polygalacturonase production by the thermophilic Bacillus sp. SMIA-2 cultivated in liquid cultures containing 0.5% (w/v apple pectin and supplemented with 0.3% (w/v corn steep liquor, reached its maximum after 36 hours with levels of 39 U.mL-1. The increase in apple pectin and corn steep liquor concentrations in the medium from 0.5 and 0.3%, respectively, to 0.65%, markedly affected the production of polygalacturonase, whose activity increased four times, reaching a maximum of 150.3 U.mL-1. Studies on polygalacturonase characterization revealed that the optimum temperature of this enzyme was between 60-70 °C. Thermostability profile indicated that the enzyme retained about 82 and 63% of its activity at 60 and 70 °C, respectively, after 2 hours of incubation. The optimum pH of the enzyme was found to be 10.0. After incubation of crude enzyme solution at room temperature for 2 hours at pH 8.0, a decrease of about 29% on its original activity was observed. At pH 10.0, the decrease was 25%.

  7. Differences in alpha 2u-globulins increased in male rat kidneys following treatment with several alpha 2u-globulin accumulating agents: cystein protease(s) play(s) an important role in production of kidney-type-alpha 2u-globulin.

    Science.gov (United States)

    Saito, K; Kaneko, H; Isobe, N; Nakatsuka, I; Yoshitake, A; Yamada, H

    1992-11-30

    Effects of alpha 2u-globulin accumulating agents on alpha 2u-globulins in rat kidneys were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting analysis. Treatment of male animals with decalin (150 mg/kg), 2,2,4-trimethylpentane (50 mg/kg), isophorone (150 mg/kg), d-limonene (150 mg/kg) or 1,4-dichlorobenzene (150 mg/kg) by gavage for 14 consecutive days in each case resulted in a marked intensification of a protein band corresponding to kidney-type-alpha 2u-globulin, with a molecular mass calculated to be approximately 16 kDa. However, intraperitoneal treatment with leupeptin and E-64 (two times 0.07 mmol/kg, for each), well known cystein protease inhibitors, while only slightly increasing this kidney-type-alpha 2u-globulin band, caused the intensification of a approximately 19-kDa molecular mass protein band which was revealed to be a native-type-alpha 2u-globulin by SDS-PAGE and immunoblotting. These results indicated that at least two types of alpha 2u-globulin can be increased in male rat kidney by chemical treatment. Moreover, cystein protease(s) appear(s) to play an important role in the degradation of alpha 2u-globulin and particularly in the conversion of native-type-alpha 2u-globulin to kidney-type-alpha 2u-globulin in rat kidneys.

  8. Enterotoxins and emetic toxins production by Bacillus cereus and other species of Bacillus isolated from Soumbala and Bikalga, African alkaline fermented food condiments.

    Science.gov (United States)

    Ouoba, Labia Irene I; Thorsen, Line; Varnam, Alan H

    2008-06-10

    The ability of various species of Bacillus from fermented seeds of Parkia biglobosa known as African locust bean (Soumbala) and fermented seeds of Hibiscus sabdariffa (Bikalga) was investigated. The study included screening of the isolates by haemolysis on blood agar, detection of toxins in broth and during the fermentation of African locust bean using the Bacillus cereus Enterotoxin Reverse Passive Latex Agglutination test kit (BCET-RPLA) and the Bacillus Diarrhoeal Enterotoxin Visual Immunoassay (BDEVIA). Detection of genes encoding cytotoxin K (CytK), haemolysin BL (Hbl A, Hbl C, Hbl D), non-hemolytic enterotoxin (NheA, NheB, NheC) and EM1 specific of emetic toxin producers was also investigated using PCR with single pair and multiplex primers. Of 41 isolates, 29 Bacillus belonging to the species of B. cereus, Bacillus subtilis, Bacillus licheniformis and Bacillus pumilus showed haemolysis on blood agar. Using RPLA, enterotoxin production was detected for three isolates of B. cereus in broth and all B. cereus (9) in fermented seeds. Using BDEVIA, enterotoxin production was detected in broth as well as in fermented seeds for all B. cereus isolates. None of the isolates belonging to the other Bacillus species was able to produce enterotoxins either by RPLA or BDEVIA. Nhe genes were detected in all B. cereus while Hbl and CytK genes were detected respectively in five and six B. cereus strains. A weak presence of Hbl (A, D) and CytK genes was detected in two isolates of B. subtilis and one of B. licheniformis but results were inconsistent, especially for Hbl genes. The emetic specific gene fragment EM1 was not detected in any of the isolates studied.

  9. 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.

  10. Chemical characterization of insoluble macromolecules (kerogen) from marine and lacustrine sediments. Molecular distribution of aliphatic dicarboxylic acids in alkaline KMnO4 oxidation products; Kaiyo oyobi konuma taisekibutsuchu no fuyosei yukibutsu (kerogen) no characterization

    Energy Technology Data Exchange (ETDEWEB)

    Morinaga, S.; Ishiwatari, R.; Machihara, T. [The University of Tokyo, Tokyo (Japan). Faculty of Science

    1996-04-15

    The insoluble macromolecules (kerogen) in marine and lacustrine sediments was oxidated by alkaline KMnO4, and the kerogen was characterized by molecular distribution of produced aliphatic dicarboxylic acids. Seven seas and ten lakes in the world are used as a sample. The oxidation products consist of n-C4-C15 {alpha}, {omega}-dicarboxylic acids, n-C7-C28 mono-carboxylic acids, and benzene carboxylic acids. The dicarboxylic acid decreases from C4 to C15 for the marine kerogen and has a maximum at C8 to C9 for the lacustrine kerogen. The method for representing the molecular distribution by two mathematical expressions was proposed. The marine and lacustrine kerogens are classified into different groups, and the sample of an eruption bay is located between the two kerogens. The polymerized structure of polyhydoroxy acids in these kerogens is assumed to have been transformed into aliphatic dicarboxylic acids by oxidation. This is also backed up by the result of cutin oxidation. 38 refs., 4 figs., 4 tags.

  11. Structure and mechanism of rhomboid protease.

    Science.gov (United States)

    Ha, Ya; Akiyama, Yoshinori; Xue, Yi

    2013-05-31

    Rhomboid protease was first discovered in Drosophila. Mutation of the fly gene interfered with growth factor signaling and produced a characteristic phenotype of a pointed head skeleton. The name rhomboid has since been widely used to describe a large family of related membrane proteins that have diverse biological functions but share a common catalytic core domain composed of six membrane-spanning segments. Most rhomboid proteases cleave membrane protein substrates near the N terminus of their transmembrane domains. How these proteases function within the confines of the membrane is not completely understood. Recent progress in crystallographic analysis of the Escherichia coli rhomboid protease GlpG in complex with inhibitors has provided new insights into the catalytic mechanism of the protease and its conformational change. Improved biochemical assays have also identified a substrate sequence motif that is specifically recognized by many rhomboid proteases.

  12. Hemisphaericin-D, a dialysable and polymerizable protease found in Bromelia hemisphaerica.

    Science.gov (United States)

    Agundis, C; Reyes, M; Córdoba, F

    1977-07-15

    Proteolytic activity was detected outside dialysis bag filled with Bromelia hemisphaerica fruit juice. The dialysable protease was concentrated and purified from small molecular weight contaminants on Sephadex G-10 columns. Acrylamide gel electrophoresis of the dialysable protease, in the presence of SDS and 2-mercaptoethanol, demonstrated a single protein band of about 8000 daltons mol. wt. The same single band with identical mobility was shown with Hemisphaericin, the enzyme retained inside the dialysis bag. The small protease, named Hemisphaericin-D was antigenic in rabbits and the antibodies cross-reacted fully with Hemisphaericin. Hemisphaericin-D appears not to be a degradation product of Hemisphaericin.

  13. Identification of the recognition sequence and target proteins for DJ-1 protease

    OpenAIRE

    Mitsugi, Hitomi; Niki, Takeshi; Takahashi-Niki, Kazuko; Tanimura, Kyoko; Yoshizawa-Kumagaye, Kumiko; Tsunemi, Masahiko; Iguchi-Ariga, Sanae M. M.; Ariga, Hiroyoshi

    2013-01-01

    DJ-1, the product of familial Parkinson's disease gene and an oncogene, is a cysteine protease which plays a role in anti-oxidative stress reaction. In this study, we identified the recognition sequence for DJ-1 protease by using recombinant DJ-1 and a peptide library. Protease activity of DJ-1 lacking C-terminal alpha-helix (DJ-1 Delta H9) was stronger than that of full-sized DJ-1, and the most susceptible sequence digested by DJ-1 Delta H9 was valine-lysine-valine-alanine (VKVA) under the o...

  14. 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.

  15. Mass spectrometry-assisted protease substrate screening

    DEFF Research Database (Denmark)

    Schlüter, Hartmut; Rykl, Jana; Thiemann, Joachim;

    2007-01-01

    Since sequencing of the human genome was completed, more than 500 genes have been annotated as proteases. Exploring the physiological role of each protease requires the identification of their natural substrates. However, the endogenous substrates of many of the human proteases are as yet unknown....... Here we describe a new assay that addresses this problem. The assay, which easily can be automated, is based on the incubation of immobilized protein fractions, which may contain the natural substrate, with a defined protease. After concentrating the proteolytically released peptides by reversed...

  16. Excretory bladder: the source of cysteine proteases in Paragonimus westermani metacercariae

    OpenAIRE

    Yang, Hyun-Jong; Chung, Young-Bae; Kang, Shin-Yong; Kong, Yoon; Cho, Seung-Yull

    2002-01-01

    The cysteine proteases of Paragonimus westermani metacercariae are involved in metacercarial excystment, host immune modulation, and possibly in tissue penetration. In order to clarify the origin of the enzymes, 28 and 27 kDa cysteine proteases in metacercarial excretory-secretory products were purified through the FPLC system using Mono Q column chromatography. The polyclonal antibodies to the enzymes were produced in BALB/c mice. Immunolocalization studies revealed that both cysteine protea...

  17. Single Cell Analysis of Leukocyte Protease Activity Using Integrated Continuous-Flow Microfluidics.

    Science.gov (United States)

    Jing, Tengyang; Lai, Zhangxing; Wu, Lidan; Han, Jongyoon; Lim, Chwee Teck; Chen, Chia-Hung

    2016-12-06

    Leukocytes are the essential cells of the immune system that protect the human body against bacteria, viruses, and other foreign invaders. Secretory products of individual leukocytes, such as matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinase (ADAMs), are critical for regulating the inflammatory response and mediating host defense. Conventional single cell analytical methods, such as flow cytometry for cellular surface biomarker studies, are insufficient for performing functional assays of the protease activity of individual leukocytes. Here, an integrated continuous-flow microfluidic assay is developed to effectively detect secretory protease activity of individual viable leukocytes. Leukocytes in blood are first washed on-chip with defined buffer to remove background activity, followed by encapsulating individual leukocytes with protease sensors in water-in-oil droplets and incubating for 1 h to measure protease secretion. With this design, single leukocyte protease profiles under naive and phorbol 12-myristate 13-acetate (PMA)-stimulated conditions are reliably measured. It is found that PMA treatment not only elevates the average protease activity level but also reduces the cellular heterogeneity in protease secretion, which is important in understanding immune capability and the disease condition of individual patients.

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

    Science.gov (United States)

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

    2009-04-01

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

  19. 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.

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

    Science.gov (United States)

    Poommarinvarakul, Sukhum; Tattiyakul, Jirarat; Muangnapoh, Chirakarn

    2010-06-01

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

  1. Production of microbial enzymes using brewers spent grain

    Energy Technology Data Exchange (ETDEWEB)

    Okita, H.; Yamashita, H.; Yabuuchi, S.

    1985-01-01

    Brewers spent grain was used as the raw material for production of three extracellular enzymes, ..cap alpha..-amylase, alkaline protease, and lichenase, by Bacillus licheniformis. The basal medium was composed of wet spent grain (about 80% moisture) and 40 mM phosphate buffer, pH 7.0 (1:5-10, w/v). For ..cap alpha..-amylase production by strain No. 18, 0.5% corn oil was added to the basal medium, and the maximum activity (66 units/ml) was found in the culture solution of the 4th day of incubation at 45 degrees C. Alkaline protease from strain H-9 was produced in the basal medium supplemented with 2 mM CaCL/sub 2/ and 10mM sodium citrate. The maximum activity during incubation at 35 degrees C was 7.2 units/ml on the 4th day. On the other hand, lichenase was produced sufficiently in the basal medium by strain Y-25, and the activity reached the level of 20 units/ml on the 3rd day of incubation at 30 degrees C. This enzyme preparation could also hydrolyze ..beta..-glucans from malt and beer. 15 references.

  2. A cyclic peptidic serine protease inhibitor

    DEFF Research Database (Denmark)

    Zhao, Baoyu; Xu, Peng; Jiang, Longguang;

    2014-01-01

    Peptides are attracting increasing interest as protease inhibitors. Here, we demonstrate a new inhibitory mechanism and a new type of exosite interactions for a phage-displayed peptide library-derived competitive inhibitor, mupain-1 (CPAYSRYLDC), of the serine protease murine urokinase...

  3. 蛋白酶在乙醇溶液中的性质及构象%The Properties and Conformation of Proteases in Ethanol Solution

    Institute of Scientific and Technical Information of China (English)

    刘玉芳; 王淼

    2011-01-01

    对碱性蛋白酶和木瓜蛋白酶在乙醇溶液中的性质和构象进行了研究。研究结果表明,在乙醇溶液中,碱性蛋白酶和木瓜蛋白酶催化反应的最适温度有不同程度下降,下降幅度为5-10℃;它们的最适pH与在缓冲液体系中相差不大。2种蛋白酶在醇溶液中的稳定性随着乙醇浓度的升高而下降,特别是当乙醇浓度上升至60%,2种蛋白酶的稳定性均迅速下降。在相同的乙醇浓度下,木瓜蛋白酶的稳定性比碱性蛋白酶高。当乙醇浓度为60%时,木瓜蛋白酶的半衰期是碱性蛋白酶的21倍。圆二色谱研究表明,在乙醇溶液中碱性蛋白酶和木瓜蛋白酶的Z-级结构均发生了明显变化,荧光光谱表明,在乙醇溶液中2种蛋白酶的荧光强度均有明显减弱,表明它们的三级结构均发生明显变化。%The properties and conformation of alkaline protease and papain in ethanol solutions were researched. The results showed that the optimum temperature of alkaline protease and papain in ethanol solutions were reduced with the increase of ethanol concentration. The range of temperature descent was 5 - 10℃. The optimum pH in ethanol solutions had little difference comparing with buffer solution. With the increase of ethanol concentration, the stability of alkaline protease and papain were affected. Especially, when the concentration of ethanol solutions was up to 60% , the stability of alkaline protease and papain decreased rapidly. The stability of papain was higher than alkaline protease in the same ethanol concentration and the half-life of papain was 21 times of alkaline protease in 60% etha- nol. The Circular dichroism spectra of the protease in ethanol solutions showed that the secondary structure of alkaline protease and papain were altered seriously. The Fluorescence spectra of the protease ethanol solution indicated that the fluorescence intensity of alkaline protease and papain in ethanol

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

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

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

  5. Protease-degradable electrospun fibrous hydrogels

    Science.gov (United States)

    Wade, Ryan J.; Bassin, Ethan J.; Rodell, Christopher B.; Burdick, Jason A.

    2015-03-01

    Electrospun nanofibres are promising in biomedical applications to replicate features of the natural extracellular matrix (ECM). However, nearly all electrospun scaffolds are either non-degradable or degrade hydrolytically, whereas natural ECM degrades proteolytically, often through matrix metalloproteinases. Here we synthesize reactive macromers that contain protease-cleavable and fluorescent peptides and are able to form both isotropic hydrogels and electrospun fibrous hydrogels through a photoinitiated polymerization. These biomimetic scaffolds are susceptible to protease-mediated cleavage in vitro in a protease dose-dependent manner and in vivo in a subcutaneous mouse model using transdermal fluorescent imaging to monitor degradation. Importantly, materials containing an alternate and non-protease-cleavable peptide sequence are stable in both in vitro and in vivo settings. To illustrate the specificity in degradation, scaffolds with mixed fibre populations support selective fibre degradation based on individual fibre degradability. Overall, this represents a novel biomimetic approach to generate protease-sensitive fibrous scaffolds for biomedical applications.

  6. Inhibition of human lymphocyte proliferation and cleavage of interleukin-2 by Pseudomonas aeruginosa proteases

    DEFF Research Database (Denmark)

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

    1988-01-01

    This study was undertaken to determine the effect of Pseudomonas aeruginosa alkaline protease (AP) and elastase (ELA) on human lymphocyte function. AP at 50 micrograms/ml and ELA at 12 micrograms/ml caused a 50% inhibition of phytohemagglutinin-induced proliferation. There was no difference......, the inhibition was partly reversed. ELA at 10 micrograms/ml cleaved IL-2, as judged by size chromatography of a reaction mixture containing 125I-labeled IL-2 and the proteases. The ELA-digested IL-2 exhibited a reduced binding capacity to IL-2 receptors on the lymphocytes. Furthermore, treatment...... of phytohemagglutinin-stimulated lymphocytes with AP and ELA resulted in inhibition of binding of intact IL-2 to IL-2 receptors on the stimulated lymphocytes. These results indicated that P. aeruginosa-derived enzymes are able to interfere with human lymphocyte function in vitro and that this effect might be due...

  7. Spatial Distribution and Speciation of Selenium in Alkaline Soils and Agricultural Products of Lanzhou%兰州碱性土壤与农产品中硒分布及形态研究

    Institute of Scientific and Technical Information of China (English)

    李伟; 李飞; 毕德; 王华魁; 李海勇; 周守标; 赵其国; 尹雪斌

    2012-01-01

    To know the spatial distribution of soil total Se and Se speciation in agricultural products, totally 473 Lanzhou soils and 122 agricultural products were sampled and analyzed. The results showed that the average total Se concentration of the topsoil in Lanzhou was (0.179± 0.066) mg/kg (n=473), which ranked in middle level of China. Soil pH value was 8.39+0.26. Soil Se varied significantly among sampling sites, and the highest averaged soil Se was found in Yuzhong Area. The bioavailable Se of soils ranged from 4.63% to15.8%, averagely 8.27% of the total Se. A significantly positive correlation between bioavilable Se and toal Se in soils was observed. For the agricultural products, the total Se in rose (Rosa rugosa), Chinese wolfberry (Lycium barbarum), broccoli (Brassica oleracea var.italica) and celery (Apium graveolens) exceeded 0.01S mg/kg (a standard value for Se-enriched products in China). The organic Se covered averagely 73.4% of the total Se in these agricultural products. Generally, the areas with higher soil Se in Lanzhou could grow higher Se products. Because of alkaline properties of Lanzhou soil, it has a significant advantage in developing Se biofortiflcation agricultural products.%通过对兰州市主要农业区表层土壤总硒有效硒调查和部分农产品总硒有机硒测定,研究了兰州市碱性农田土壤与农产品中硒的积累特征和分布规律.结果表明,兰州市农田表层土壤pH 8.39±0.26,总硒含量为(0.179±0.066) mg/kg(n=473),处于全国中等水平.全市土壤总硒含量区域性差别较大,市辖区明显低于三县,榆中县居全市之首.土壤有效硒与总硒含量显著正相关,有效硒占比为4.63%~15.8%,平均8.27%.农产品中玫瑰(Rosa rugosa),部分西兰花(Brassicaoleracea var.italica)和芹菜(Apium graveolens)样品硒含量高于0.015 mg/kg,为该市天然富硒农产品.农产品中有机硒占总硒比例较高,均值为73.4%.从区域整体来看,土壤硒含量较高的

  8. Atomic resolution crystal structure of Sapp2p, a secreted aspartic protease from Candida parapsilosis.

    Science.gov (United States)

    Dostál, Jiří; Pecina, Adam; Hrušková-Heidingsfeldová, Olga; Marečková, Lucie; Pichová, Iva; Řezáčová, Pavlina; Lepšík, Martin; Brynda, Jiří

    2015-12-01

    The virulence of the Candida pathogens is enhanced by the production of secreted aspartic proteases, which therefore represent possible targets for drug design. Here, the crystal structure of the secreted aspartic protease Sapp2p from Candida parapsilosis was determined. Sapp2p was isolated from its natural source and crystallized in complex with pepstatin A, a classical aspartic protease inhibitor. The atomic resolution of 0.83 Å allowed the protonation states of the active-site residues to be inferred. A detailed comparison of the structure of Sapp2p with the structure of Sapp1p, the most abundant C. parapsilosis secreted aspartic protease, was performed. The analysis, which included advanced quantum-chemical interaction-energy calculations, uncovered molecular details that allowed the experimentally observed equipotent inhibition of both isoenzymes by pepstatin A to be rationalized.

  9. 2nd Generation Alkaline Electrolysis

    DEFF Research Database (Denmark)

    Yde, Lars; Kjartansdóttir, Cecilia Kristin; Allebrod, Frank;

    This report provides the results of the 2nd Generation Alkaline Electrolysis project which was initiated in 2008. The project has been conducted from 2009-2012 by a consortium comprising Århus University Business and Social Science – Centre for Energy Technologies (CET (former HIRC)), Technical...

  10. Mast cell proteases as pharmacological targets.

    Science.gov (United States)

    Caughey, George H

    2016-05-05

    Mast cells are rich in proteases, which are the major proteins of intracellular granules and are released with histamine and heparin by activated cells. Most of these proteases are active in the granule as well as outside of the mast cell when secreted, and can cleave targets near degranulating mast cells and in adjoining tissue compartments. Some proteases released from mast cells reach the bloodstream and may have far-reaching actions. In terms of relative amounts, the major mast cell proteases include the tryptases, chymases, cathepsin G, carboxypeptidase A3, dipeptidylpeptidase I/cathepsin C, and cathepsins L and S. Some mast cells also produce granzyme B, plasminogen activators, and matrix metalloproteinases. Tryptases and chymases are almost entirely mast cell-specific, whereas other proteases, such as cathepsins G, C, and L are expressed by a variety of inflammatory cells. Carboxypeptidase A3 expression is a property shared by basophils and mast cells. Other proteases, such as mastins, are largely basophil-specific, although human basophils are protease-deficient compared with their murine counterparts. The major classes of mast cell proteases have been targeted for development of therapeutic inhibitors. Also, a human β-tryptase has been proposed as a potential drug itself, to inactivate of snake venins. Diseases linked to mast cell proteases include allergic diseases, such as asthma, eczema, and anaphylaxis, but also include non-allergic diseases such as inflammatory bowel disease, autoimmune arthritis, atherosclerosis, aortic aneurysms, hypertension, myocardial infarction, heart failure, pulmonary hypertension and scarring diseases of lungs and other organs. In some cases, studies performed in mouse models suggest protective or homeostatic roles for specific proteases (or groups of proteases) in infections by bacteria, worms and other parasites, and even in allergic inflammation. At the same time, a clearer picture has emerged of differences in the

  11. Identification of a Bacterium Isolated from the Decayed Cyanobacteria and Its Fermentation for Protease Production%一株从腐烂蓝藻中分离的细菌的鉴定及其发酵产蛋白酶研究

    Institute of Scientific and Technical Information of China (English)

    尹静; 蔡宇杰; 廖祥儒; 郝之奎; 李婷婷; 李桂英; 张大兵

    2011-01-01

    应用形态学、生理生化和16S rRNA基因序列分析方法,鉴定了从腐烂蓝藻中分离得到的一株细菌,利用液态发酵法探讨了碳、氮源等对其发酵产蛋白酶的影响并研究了其部分酶学性质.结果表明,该菌可归入沙雷氏菌属,定名为Serratia marcescens SYBC YH,其最适碳源为果糖、葡萄糖、羽毛粉或玉米粉,最适氮源为玉米浆粉、酵母膏和牛肉膏;该菌还可以蓝藻为唯一氮源发酵产耐有机溶剂蛋白酶.该酶的最适温度为40℃,最适pH为7.0.%Morphological aspects, physiological and biochemical characters and 16 S rRNA sequence were utilized to identify the bacterium isolated from the decayed cyanobacteria in this study, and liquid state fermentation was utilized to evaluate the effect of carbon and nitrogen resources on its protease production. The data showed that the bacterium isolated was belonged to the genus Serratia designated as Serratia marcescens SYBC YH. The optimal carbon resource was fructose, glucose, feather powder, or powder of corn seed. The optimal nitrogen resource was corn steep powder, yeast extract or beef extract. The strain could produce protease by liquid state fermentation with cyanobacteria as sole nitrogen resource to produce organic solvent tolerant protease. The optimal catalytic temperature and pH was 40°C and 7. 0 respectively.

  12. Caspase Family Proteases and Apoptosis

    Institute of Scientific and Technical Information of China (English)

    Ting-Jun FAN; Li-Hui HAN; Ri-Shan CONG; Jin LIANG

    2005-01-01

    Apoptosis, or programmed cell death, is an essential physiological process that plays a critical role in development and tissue homeostasis. The progress of apoptosis is regulated in an orderly way by a series of signal cascades under certain circumstances. The caspase-cascade system plays vital roles in the induction, transduction and amplification of intracellular apoptotic signals. Caspases, closely associated with apoptosis, are aspartate-specific cysteine proteases and members of the interleukin-1β-converting enzyme family. The activation and function of caspases, involved in the delicate caspase-cascade system, are regulated by various kinds of molecules, such as the inhibitor of apoptosis protein, Bcl-2 family proteins, calpain,and Ca2+. Based on the latest research, the members of the caspase family, caspase-cascade system and caspase-regulating molecules involved in apoptosis are reviewed.

  13. An investigation into the influences of species and biotype on the type of IgA1 protease produced by isolates of Haemophilus.

    Science.gov (United States)

    Senior, B W; Ip, C L

    1999-04-01

    A total of 59 isolates of different Haemophilus spp., mostly from clinical specimens, was characterised, biotyped and examined for production of type 1 or type 2 IgA1 protease. IgA1 protease activity was not found in any isolate of a species with no or low virulence for man including H. parainfluenzae, H. haemolyticus, H. aphrophilus, H. paraphrophilus, H. segnis, H. paraphrohaemolyticus and H. haemoglobinophilus. IgA1 protease was produced by all isolates of H. influenzae and H. aegyptius and by some isolates of H. parahaemolyticus. The type of IgA1 protease appeared to be independent of the biotype of the isolate in H. influenzae. For the first time some isolates of H. aegyptius were found that produced type 2 IgA1 protease. IgA1 protease production in H. parahaemolyticus may be associated with the virulence of the isolate.

  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. Analysis of the proteases activity in 13 organs of the doll fish (Andrias davidianus) digestive system%中国大鲵消化系统13种器官的蛋白水解酶种类和活性分析

    Institute of Scientific and Technical Information of China (English)

    乔志刚; 辛泽华; 李吉学; 沈国民; 徐存拴

    2003-01-01

    Protease activity in 13 doll fish, Andrias davidianus, digestive organs: tongue, esophagus, cardia, stomach,pylorus, duodenum, jejunum, ileum, rectum, cloacal chamber, liver, pancreas, gall bladder were analysed with gelatine polyacrylamide gel-electrophoresis (G-PAGE). Compared to rats and rhesus monkeys (Macaca mullatta tcheliensis ),protease activity in the Andrias davidianus digestive system was more varied and stronger. The activity of acid proteases>that of the neutral proteases>that of the alkaline proteases. The kinds and activities of neutral and alkaline proteases in the Andrias davidianus digestive system wereare very different, while the kinds of acid proteases in them were very simi-lar, but their activities are different. Analysing the activities of proteases in these organs shows that protease activity in the duodenum > that in rectum > that in stomach > that in pancreas > that in liver. Experimental results suggest that the function of the rectum and liver in Andrias daviclianus may be as important as that of the duodenum and pancreas in food degradation.

  16. Specificity of proteinase K at P2 to P3' sub-sites and its comparison to other serine proteases.

    Science.gov (United States)

    Qasim, Mohammad A

    2014-01-01

    Specificity of the commercially important serine protease, proteinase K, has been investigated by measuring free energies of association of proteinase K with turkey ovomucoid third domain inhibitor variants at contact positions P2, P1, P1', P2', and P3'. Correlations of these values were run with similar values that have been obtained for six other serine proteases. Among the six proteases, subtilisin Carlsberg shows a near perfect correlation (Pearson Product correlation coefficient = 0.93 to 0.99) with proteinase K at all of these positions. Proteinase K has only 35% sequence identity with subtilisin Carlsberg, yet, the two enzymes are nearly identical in their specificity at P2 to P3' positions. With other serine proteases such as bovine chymotrypsin, human leukocyte elastase, porcine pancreatic elastase, Streptomyces griseus protease A and B, proteinase K showed relatively poor or no correlation.

  17. Alkalinity and hardness: Critical but elusive concepts in aquaculture

    Science.gov (United States)

    Total alkalinity and total hardness are familiar variables to those involved in aquatic animal production. Aquaculturists – both scientists and practitioners alike – tend to have some understanding of the two variables and of methods for adjusting their concentrations. The chemistry and the biolog...

  18. Evaluation of some bean lines tolerance to alkaline soil

    Directory of Open Access Journals (Sweden)

    Abeer A. Radi

    2012-01-01

    Full Text Available Introduction: In less arid climates, salts are less concentrated and sodium dominates in carbonate and bicarbonate forms, which enhance the formation of alkaline soils. The development and identification of salt-tolerant crop cultivars or lines would complement salt management programs to improve the productivity and yields of salt stressed plants.Materials and methods: This work was to study the evaluation of alkalinity tolerance of some bean lines grown under different levels of sodium carbonate (Na2CO3 to select the most alkalinity tolerant lines versus the most-sensitive ones out of 6 lines of the test plants.Results: The symptoms induced by alkalinity included reduction in root, shoot growth, and leaf area which were more severe in some bean lines. Potassium leakage was severely affected by alkalinity in some lines at all tested levels, while in some others a moderate damage was manifested only at the higher levels. The increase in Na2CO3 level was associated with a gradual fall in chlorophyll a and b biosynthesis of all the test bean lines. However, alkalinity at low and moderate levels had a favorable effect on the biosynthesis of carotenoids in all the test bean lines. The increase in Na2CO3 supply had a considerable stimulatory effect on sodium accumulation, while potassium accumulation fluctuated in organs of bean lines.Conclusion: Assiut 1104 out of all the different lines investigated was found to display the lowest sensitivity to alkalinity stress, while Assiut 12/104 was the most sensitive one.

  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. Immobilization of cesium in alkaline activated fly ash matrix

    Science.gov (United States)

    Fernandez-Jimenez, A.; Macphee, D. E.; Lachowski, E. E.; Palomo, A.

    2005-11-01

    The immobilization potential of alkaline activated fly ash (AAFA) matrices for cesium has been investigated. The presence of Cs in the AAFA pastes, prepared using 8M NaOH solution as activator, showed no significant adverse effects on mechanical strength or microstructure, nor were significant quantities of Cs leached following application of the Toxic Characteristic Leaching Procedure (TCLP) and American Nuclear Society (ANS) 16.1 leaching protocols. Microstructural analysis shows Cs associated with the main reaction product in the AAFA suggesting that cesium is chemically bound rather than physically encapsulated. It is proposed that cesium is incorporated into the alkaline aluminosilicate gel, a precursor for zeolite formation.

  1. Ahp cyclodepsipeptides: the impact of the Ahp residue on the "canonical inhibition" of S1 serine proteases.

    Science.gov (United States)

    Stolze, Sara C; Meltzer, Michael; Ehrmann, Michael; Kaiser, Markus

    2013-07-22

    S1 serine proteases are by far the largest and most diverse family of proteases encoded in the human genome. Although recent decades have seen an enormous increase in our knowledge, the biological functions of most of these proteases remain to be elucidated. Chemical inhibitors have proven to be versatile tools for studying the functions of proteases, but this approach is hampered by the limited availability of inhibitor scaffold structures with the potential to allow rapid discovery of selective, noncovalent small-molecule protease inhibitors. The natural product class of Ahp cyclodepsipeptides is an unusual class of small-molecule canonical inhibitors; the incorporation of protease cleavage sequences into their molecular scaffolds enables the design of specific small-molecule inhibitors that simultaneously target the S and S' subsites of the protease through noncovalent mechanisms. Their synthesis is tedious, however, so in this study we have investigated the relevance of the Ahp moiety for achieving potent inhibition. We found that although the Ahp residue plays an important role in inhibition potency, appropriate replacement with β-hydroxy amino acids results in structurally less complex derivatives that inhibit serine proteases in the low micromolar range.

  2. The role of proteases in the differentiation of Acanthamoeba castellanii.

    Science.gov (United States)

    Dudley, Ricky; Alsam, Selwa; Khan, Naveed Ahmed

    2008-09-01

    Proteases are significant determinants of protozoan pathogenicity and cytolysis of host cells. However, there is now growing evidence of their involvement in cellular differentiation. Acanthamoeba castellanii of the T4 genotype elaborates a number of proteases, which are inhibited by the serine protease inhibitor phenylmethylsulphonyl fluoride. Using this and other selective protease inhibitors, in tandem with siRNA primers, specific to the catalytic site of Acanthamoeba serine proteases, we demonstrate that serine protease activity is crucial for the differentiation of A. castellanii. Furthermore, both encystment and excystment of A. castellanii was found to be dependent on serine protease function.

  3. Development of alkaline fuel cells.

    Energy Technology Data Exchange (ETDEWEB)

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

    2013-09-01

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