Search results for: cross-linked enzyme aggregates

Authors: Yasser Gaber, Mohamed Ismail, Serena Bisagni, Mohamad Takwa, Rajni Hatti-Kaul

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Enzymes are safe and selective catalysts. They skillfully catalyze chemical reactions; however, the native form is not usually suitable for industrial applications. Enzymes are therefore engineered by several techniques to meet the required catalytic task. Clopidogrel is recorded among the five best selling pharmaceutical in 2010 under the brand name Plavix. The commonly used route for production of the drug on an industrial scale is the synthesis of the racemic mixture followed by diastereomeric resolution to obtain the pure S isomer. The process consumes a lot of solvents and chemicals. We have evaluated a biocatalytic cleaner approach for asymmetric hydrolysis of racemic clopidogrel. Initial screening of a selected number of hydrolases showed only one enzyme EST to exhibit activity and selectivity towards the desired stereoisomer. As the crude EST is a mixture of several isoenzymes, a homology model of EST-1 was used in molecular dynamic simulations to study the interaction of the enzyme with R and S isomers of clopidogrel. Analysis of the geometric hindrances of the tetrahedral intermediates revealed a potential site for mutagenesis in order to improve the activity and the selectivity. Single point mutation showed dramatic increase in activity and inversion of the enantioselectivity (400 fold change in E value).

Keywords: biocatalysis, biotechnology, enzyme, protein engineering, molecular modeling

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Authors: A. K. M. Kafi, S. N. Nina, Mashitah M. Yusoff

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In this work, we have described a new 3-dimensional (3D) network of cross-linked Horseradish Peroxidase/Carbon Nanotube (HRP/CNT) on a thiol-modified Au surface in order to build up the effective electrical wiring of the enzyme units with the electrode. This was achieved by the electropolymerization of aniline-functionalized carbon nanotubes (CNTs) and 4-aminothiophenol -modified-HRP on a 4-aminothiophenol monolayer-modified Au electrode. The synthesized 3D HRP/CNT networks were characterized with cyclic voltammetry and amperometry, resulting the establishment direct electron transfer between the redox active unit of HRP and the Au surface. Electrochemical measurements reveal that the immobilized HRP exhibits high biological activity and stability and a quasi-reversible redox peak of the redox center of HRP was observed at about −0.355 and −0.275 V vs. Ag/AgCl. The electron transfer rate constant, KS and electron transfer co-efficient were found to be 0.57 s-1 and 0.42, respectively. Based on the electrocatalytic process by direct electrochemistry of HRP, a biosensor for detecting H2O2 was developed. The developed biosensor exhibits excellent electrocatalytic activity for the reduction of H2O2. The proposed biosensor modified with HRP/CNT 3D network displays a broader linear range and a lower detection limit for H2O2 determination. The linear range is from 1.0×10−7 to 1.2×10−4M with a detection limit of 2.2.0×10−8M at 3σ. Moreover, this biosensor exhibits very high sensitivity, good reproducibility and long-time stability. In summary, ease of fabrication, a low cost, fast response and high sensitivity are the main advantages of the new biosensor proposed in this study. These obvious advantages would really help for the real analytical applicability of the proposed biosensor.

Keywords: redox enzyme, nanomaterials, biosensors, electrical communication

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Authors: Salahaldein Alsadey, Issa Amaish

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Recycled aggregates (RA) have the potential to compromise concrete performance, contributing to issues such as reduced strength and increased susceptibility to cracking. This study investigates the impact of sisal fiber (SF) on the mechanical properties of concrete, with the objective of utilizing sisal fibers as a reinforcing element in concrete compositions containing natural aggregate and varying percentages (25%, 50%, and 75%) of coarse recycled aggregate replacement. The investigation aims to discern the positive and negative effects on compressive and flexural strength, thereby assessing the viability of sisal fiber-reinforced recycled concrete in comparison to conventional concrete composed of natural aggregate without sisal fiber. Test results revealed that concrete samples incorporating sisal fiber exhibited elevated compressive and flexural strength. Comparative analysis of these strength values was conducted with reference to samples devoid of sisal fiber.

Keywords: sustainable construction, construction materials, recycled aggregate, sisal fibers, compressive strength, flexural strength, eco-friendly concrete, natural fiber composites, recycled materials, construction waste management

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Authors: Shegufta Zahan, Muhammad A. Zahin, Muhammad M. Hossain, Raquib Ahsan

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Rice Husk Ash (RHA) is one of the agricultural waste byproducts available widely in the world and contains a large amount of silica. In Bangladesh, stones cannot be used as coarse aggregate in infrastructure works as they are not available and need to be imported from abroad. As a result, bricks are mostly used as coarse aggregates in concrete as they are cheaper and easily produced here. Clay is the raw material for producing brick. Due to rapid urban growth and the industrial revolution, demand for brick is increasing, which led to a decrease in the topsoil. This study aims to produce lightweight block aggregates with sufficient strength utilizing RHA at low cost and use them as an ingredient of concrete. RHA, because of its pozzolanic behavior, can be utilized to produce better quality block aggregates at lower cost, replacing clay content in the bricks. The whole study can be divided into three parts. In the first part, characterization tests on RHA and clay were performed to determine their properties. Six different types of RHA from different mills were characterized by XRD and SEM analysis. Their fineness was determined by conducting a fineness test. The result of XRD confirmed the amorphous state of RHA. The characterization test for clay identifies the sample as “silty clay” with a specific gravity of 2.59 and 14% optimum moisture content. In the second part, blocks were produced with six different types of RHA with different combinations by volume with clay. Then mixtures were manually compacted in molds before subjecting them to oven drying at 120 °C for 7 days. After that, dried blocks were placed in a furnace at 1200 °C to produce ultimate blocks. Loss on ignition test, apparent density test, crushing strength test, efflorescence test, and absorption test were conducted on the blocks to compare their performance with the bricks. For 40% of RHA, the crushing strength result was found 60 MPa, where crushing strength for brick was observed 48.1 MPa. In the third part, the crushed blocks were used as coarse aggregate in concrete cylinders and compared them with brick concrete cylinders. Specimens were cured for 7 days and 28 days. The highest compressive strength of block cylinders for 7 days curing was calculated as 26.1 MPa, whereas, for 28 days curing, it was found 34 MPa. On the other hand, for brick cylinders, the value of compressing strength of 7 days and 28 days curing was observed as 20 MPa and 30 MPa, respectively. These research findings can help with the increasing demand for topsoil of the earth, and also turn a waste product into a valuable one.

Keywords: characterization, furnace, pozzolanic behavior, rice husk ash

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Authors: Salina Y. Saddick

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Mild gestational hyperglycemia (MGH) is a very common complication of pregnancy that is characterized by intolerance to glucose. The association of angiotensin-converting enzyme (ACE) insertion/deletion (I/D) polymorphism to MGH has been previously reported. In this study, we evaluated the association between ACE polymorphism and the risk of MGH in a Saudi population. We conducted a case-control study in a population of 100 MGH patients and 100 control subjects. ACE gene polymorphism was analyzed by the novel approach of tetraprimer amplification refractory mutation system (ARMS)-polymerase chain reaction (PCR). The frequency of ACE polymorphism was not associated with either alleles or genotypes in MGH patients. Glucose concentration was found to be significantly associated with the MGH group. Our study suggests that ACE genotypes were not associated with ACE polymorphism in a Saudi population.

Keywords: MGH, ACE, insertion polymorphism, deletion polymorphism

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Authors: G. K. Sruthi, Sila Bhattacharya

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Sorghum is an important food crop in the dry tropical areas of the world, and possesses significant levels of phytochemicals and dietary fiber to offer health benefits. However, the absence of gluten is a limitation for converting the sorghum dough into sheeted/flattened/rolled products. Chapathi/roti (flat unleavened bread prepared conventionally from whole wheat flour dough) was attempted from sorghum as wheat gluten causes allergic reactions leading to celiac disease. Dynamic oscillatory rheology of sorghum flour dough (control sample) and enzyme treated sorghum doughs were studied and linked to the attributes of the finished ready-to-eat product. Enzymes like amylase, xylanase, and a mix of amylase and xylanase treated dough affected drastically the rheological behaviour causing a lowering of dough consistency. In the case of amylase treated dough, marked decrease of the storage modulus (G') values from 85513 Pa to 23041 Pa and loss modulus (G") values from 8304 Pa to 7370 Pa was noticed while the phase angle (δ) increased from 5.6 to 10.1o for treated doughs. There was a 2 and 3 fold increase in the total sugar content after α-amylase and xylanase treatment, respectively, with simultaneous changes in the structure of the dough and finished product. Scanning electron microscopy exhibited enhanced extent of changes in starch granules. Amylase and mixed enzyme treatment produced a sticky dough which was difficult to roll/flatten. The dough handling properties were improved by the use of xylanase and quality attributes of the chapath/roti. It is concluded that enzyme treatment can offer improved rheological status of gluten free doughs and products.

Keywords: sorghum dough, amylase, xylanase, dynamic oscillatory rheology, sensory assessment

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Authors: Mokhtar Nikgoo

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Definition of Concrete and Concreting: Concrete (in French: Béton) in a broad sense is any substance or combination that consists of a sticky substance with the property of cementation. In general, concrete refers to concrete made by Portland cement, which is produced by mixing fine and coarse aggregates, Portland cement and water. After enough time, this mixture turns into a stone-like substance. During the hardening or processing of the concrete, cement is chemically combined with water to form strong crystals that bind the aggregates together, a process called hydration. During this process, significant heat is released called hydration heat. Additionally, concrete shrinks slightly, especially as excess water evaporates, a phenomenon known as drying shrinkage. The process of hardening and the gradual increase in concrete strength that occurs with it does not end suddenly unless it is artificially interrupted. Instead, it decreases more over long periods of time, although, in practical applications, concrete is usually set after 28 days and is considered at full design strength. Concrete may be made from different types of cement as well as pozzolans, furnace slag, additives, additives, polymers, fibers, etc. It may also be used in the way it is made, heating, water vapor, autoclave, vacuum, hydraulic pressures and various condensers.

Keywords: concrete, RCC, batching, cement, Pozzolan, mixing plan

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Authors: Andreas Schomburg, Claire Le Bayon, Claire Guenat, Philip Brunner

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Numerous river restoration projects have been established in Switzerland in recent years after decades of human activity in floodplains. The success of restoration projects in terms of biodiversity and ecosystem functions largely depend on the development of the floodplain soil system. Plants and earthworms as ecosystem engineers are known to be able to build up a stable soil structure by incorporating soil organic matter into the soil matrix that creates water stable soil aggregates. Their engineering efficiency however largely depends on changing soil properties and frequent floods along an evolutive floodplain transect. This study, therefore, aims to quantify the effect of flood frequency and duration as well as of physico-chemical soil parameters on plants’ and earthworms’ engineering efficiency. It is furthermore predicted that these influences may have a different impact on one of the engineers that leads to a varying contribution to aggregate formation within the floodplain transect. Ecosystem engineers were sampled and described in three different floodplain habitats differentiated according to the evolutionary stages of the vegetation ranging from pioneer to forest vegetation in a floodplain restored 15 years ago. In addition, the same analyses were performed in an embanked adjacent pasture as a reference for the pre-restored state. Soil aggregates were collected and analyzed for their organic matter quantity and quality using Rock Eval pyrolysis. Water level and discharge measurements dating back until 2008 were used to quantify the return period of major floods. Our results show an increasing amount of water stable aggregates in soil with increasing distance to the river and show largest values in the reference site. A decreasing flood frequency and the proportion of silt and clay in the soil texture explain these findings according to F values from one way ANOVA of a fitted mixed effect model. Significantly larger amounts of labile organic matter signatures were found in soil aggregates in the forest habitat and in the reference site that indicates a larger contribution of plants to soil aggregation in these habitats compared to the pioneer vegetation zone. Earthworms’ contribution to soil aggregation does not show significant differences in the floodplain transect, but their effect could be identified even in the pioneer vegetation with its large proportion of coarse sand in the soil texture and frequent inundations. These findings indicate that ecosystem engineers seem to be able to create soil aggregates even under unfavorable soil conditions and under frequent floods. A restoration success can therefore be expected even in ecosystems with harsh soil properties and frequent external disturbances.

Keywords: ecosystem engineers, flood frequency, floodplains, river restoration, rock eval pyrolysis, soil organic matter incorporation, soil structuration

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Authors: L. L. Tundisi, A. Pessoa Jr., E. B. Tambourgi, E. Silveira, P. G. Mazzola

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L-asparaginase (L-ASNase) is the gold standard treatment for acute lymphoblastic leukemia that mainly affects pediatric patients; treatment increases survival from 20% to 90%. The characterization of other L-Asparaginases, apart from the most used from Escherichia coli and Erwinia chrysanthemi, has been reported, but the choice of the most appropriate is still under debate. This choice should be based on its pharmacokinetics, immune hypersensitivity, doses, prices, pharmacodynamics. The main factors influencing the antileukemic activity of ASNase are enzymatic activity, Km, glutaminase activity, clearance of the enzyme and development of resistance. However, most of the commercialized enzyme present an intrinsic glutaminase activity, which is responsible for some side effects. In this study, glutaminase free asparaginase produced from Aspergillus oryzae was precipitated in different percentages of ethanol (0–80%), until optimum ethanol concentration of 60% (w/w) was found. Following, precipitation of crude L-ASNase was performed in a single step, using 60% (w/w) ethanol, under constant agitation and temperature. It presented activity of 135.45 U/mg and after gel filtration chromatography with Sephadex G-the enzymatic activity was 322.02 U/mg. The apparent molecular mass of the purified L-ASNase fraction was estimated by 10% SDS-PAGE. Proteins were stained with Coomassie Brilliant Blue R-250. The molar mass range was from 10 kDa to 250 kDa. L-ASNase from Aspergillus oryzae was characterized aiming possible therapeutic use. Four different buffers (phosphate-citrate buffer pH 2.6 to 5.8; phosphate buffer pH 5.8 to 7.4; Tris - HCl pH 7.4 to 9.0; and carbonate buffer pH 9.8 to 10.6) were used to measure the optimum pH for L-ASNase activity. The optimum temperature for enzyme activity was measured at optimal pH conditions (Tris-HCl and phosphate buffer, pH 7.4) at different temperatures ranging from 5 to 55°C. All activities were calculated by quantifying the free ammonia, using the Nessler reagent. The kinetic parameters calculation, e.g. Michaelis-Menten constant (Km), maximum velocity (Vmax) and Hills coefficient (n), were performed by incubating the enzyme in different concentrations of the substrate at optimum conditions of pH and fitted on Hill’s equation. This glutaminase free asparaginase showed a low Km (3.39 mM and 3.81 mM) and enzymatic activity of 135.45 U/mg after precipitation with ethanol. After gel filtration chromatography it rose to 322.02 U/mg. Optimum activity was found between pH 5.8 - 9.0, best activity results with phosphate buffer pH 7.4 and Tris-HCl pH 7.4 and showed activity from 5°C to 55°C. These results indicate that L-ASNase from A. oryzae has the potential for human use.

Keywords: biopharmaceuticals, bioprocessing, bioproducts, biotechnology, enzyme activity, ethanol precipitation

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Authors: Haneef Ur Rehman, Afsheen Aman, Abdul Hameed Baloch, Shah Ali Ul Qader

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Pectinase is a heterogeneous group of enzymes that catalyze the hydrolysis of pectin substances and widely has been used in food and textile industries. In current study, pectinase from B. licheniformis KIBGE-IB21 was immobilized within different polymers (calcium alginate beads, polyacrylamide gel and agar-agar matrix) to enhance its catalytic properties. Polyacrylamide gel was found to be most promising one and gave maximum (89%) immobilization yield. While less immobilization yield was observed in case of calcium alginate beads that only retained 46 % activity. The reaction time for maximum pectinolytic activity was increased from 5.0 to 10 minutes after immobilization. The temperature of pectinase for maximum enzyme activity was increased from 45 °C to 50 °C and 55 °C when it was immobilized within agar-agar and calcium alginate beads, respectively. The optimum pH of pectinase didn’t alter when it was immobilized within polyacrylamide gel and calcium alginate beads, but in case of agar-agar it was changed from pH 10 to pH 9.0. Thermal stability of pectinase was improved after immobilization and immobilized pectinase showed higher toleration against different temperatures as compared to free enzyme. It can be concluded that the entrapment is a simple, single step and promising procedure to immobilized pectinase within different synthetic and non-synthetic polymers and enhanced its catalytic properties.

Keywords: pectinase, characterization immobilization, polyacrylamide, agar-agar, calcium alginate beads

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Authors: Fatima Zohra Ibn Majdoub Hassani, Ivan Lavandera, Joseph Kreit

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This study explored the effects of different organic solvents, temperature, and the amount of glycerol on the alcohol dehydrogenase (ADH)-catalysed stereoselective reduction of different ketones. These conversions were then analyzed by gas chromatography. It was found that when the amount of deep eutectic solvents (DES) increases, it can improve the stereoselectivity of the enzyme although reducing its ability to convert the substrate into the corresponding alcohol. Moreover, glycerol was found to have a strong stabilizing effect on the ADH from Ralstonia sp. (E. coli/ RasADH). In the case of organic solvents, it was observed that the best conversions into the alcohols were achieved with DMSO and hexane. It was also observed that temperature decreased the ability of the enzyme to convert the substrates into the products and also affected the selectivity. In addition to that, the recycling of DES up to three times gave good conversions and enantiomeric excess results and glycerol showed a positive effect in the stability of various ADHs. Using RasADH, a good conversion and enantiomeric excess into the S-alcohol were obtained. It was found that an enhancement of the temperature disabled the stabilizing effect of glycerol and decreased the stereoselectivity of the enzyme. However, for other ADHs a temperature increase had an opposite positive effect, especially with ADH-T from Thermoanaerobium sp. One of the objectives of this study was to see the effect of cofactors such as NAD(P) on the biocatlysis activities of ADHs.

Keywords: alcohol dehydrogenases, DES, gas chromatography, RasADH

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Authors: Salem Abdalla

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Objective: Dipyrone is a widely used, well tolerated analgesic drug which, however, is compromised by agranulocytosis as an adverse effect. Subsequent to no enzymatic hydrolysis, the primary metabolic step is N-demethylation of 4-methylaminoantipyrine (4-MAA) to 4-aminoantipyrine (4-AA). The aim of the present study was to identify the cytochrome P-450 enzyme (CYP) mediating this reaction. Methods: We identified the relevant CYP using virus expressed isolated rat liver microsomes with chemical inhibition studies. The substrate of 4-methylaminantipyrine was employed at six different concentrations (25, 50, 100, 400, 800, and 1200 µmol/l) with varying concentrations of selective inhibitors of CYP1A2 (furafylline, fluvoxamine), CYP3A4 (ketoconazole), CYP2A6 (coumarin), CYP2D6 (quinidine), CYP2C19 (omeprazole, fluvoxamine, tranylcypromine), CYP2C9 (sulfaphenazole), and CYP1A1 (alpha-naphthoflavone). 4-MAA and 4-AA were analyzed by HPLC, and enzyme kinetic parameters (Km and Vmax) were determined by regression (Sigma plot 9.0). Results: The N-demethylation of 4-MAA by microsomes prepared from baculovirus-expressing human CYP was pronounced with CYP2C19. Intrinsic clearances of the most active enzymes were 0.092, 0.027, and 0.026 for the CYP enzymes 2C19, 2D6, and 1A2, respectively. Metabolism by rat liver microsomes was strongly inhibited by omeprazole (IC50 of 0.05). Conclusion: The enzyme CYP2C19 apparently has an important role in N-demethylation of 4-methylaminoantipyrine which should be further analyzed in clinical studies and which may also be interesting concerning the agranulocytosis.

Keywords: dipyrone, 4-methylaminoantipyrine (4-MAA), 4- aminoantipyrine (4-AA), metabolism, human CYP2C19

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Authors: Hina Gul Rajpoot, Wazeer Hussain Solangi

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Experimental Design (DOE) economically maximizes information; we deliberately change one or more process variables (looms) in order to observe the effect the changes have on one or more response fabric properties. In DOE obtained data can be analyzed to yield valid and objective conclusions. An Experimental Design is lying out of a detailed experimental plan in advance and maximizes the amount of "information" that can be obtained for a given amount of experimental. Fabric of 36 inches having following weaves was used. 3/1 twill, warp cotton (10.5 den), weft Lycra (16 spandex * 70 den) Ends per inch86, Picks per inch 52 and washes process includes Stone wash, Rinse wash, Bleaching and Enzyme wash. Once the samples were ready, they were subjected to tensile and tear strength tests, for these two kinds of samples were considered. One washed fabric samples of warp direction type and other type of the samples was weft direction. Then five samples from each were considered for tensile and teat strength tests separately then takes the mean value. The results found that the lowest strength damaged in the weft direction observed by tensile strength test & Enzyme wash. Maximum breaking load of the enzyme washed fabric sample was 42 kg.

Keywords: twill, indigo dye, tear strength, loom, ball warp, denier or den, seam, waist band, pilling, selvage

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Authors: Edgar Aguilar-Ortíz, Nicolas Lévaray, Mireille Vonlanthen, Eric G. Morales-Espinoza, Ernesto Rivera, Xiao Xia Zhu

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Four new star compounds bearing a porphyrin core and cholic acid units, (TPPh(Zn) tetra-CA, TPPh(2H) tetra-CA, TPPh(Zn) octa-CA and TPPh(2H) octa-CA), have been synthesized using the Click Chemistry approach, which consist on azide-alkyne couplings. These novel functionalized porphyrins were characterized by 1H and 13C NMR spectroscopy and their structure was confirmed by MALDI-TOF. The optical properties of these compounds were studied by absorption and fluorescence spectroscopy. On the other hand, order to evaluate the amphiphilic properties of the cholic acid units combined with the optical response of the porphyrin core, we performed absorption and fluorescence studies in function of the polarity of the environment. It was found that as soon as we increase the polarity of the solvent, the Zn-metallated porphyrins, (TPPh(Zn) tetra-CA and TPPh(Zn) octa-CA), are able to form J aggregates, whereas the free-base porphyrins, TPPh(2H) tetra-CA and TPPh(2H) octa-CA, behaved differently.

Keywords: aggregates, amphiphilic, cholic acid, click-chemistry, porphyrin

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Authors: Ji Hye Im, Nguyen T. T. Phuong, Keon Wook Kang

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Estrogens are important for the development and growth of estrogen receptor (ER)-positive breast cancer, for which anti-estrogen therapy is one of the most effective treatments. However, its efficacy can be limited by either de novo or acquired resistance. Aromatase is a key enzyme for the biosynthesis of estrogens, and inhibition of this enzyme leads to profound hypoestrogenism. Here, we found that the basal expression and activity of aromatase were significantly increased in tamoxifen (TAM)-resistant human breast cancer (TAMR-MCF-7) cells compared to control MCF-7 cells. We further revealed that aromatase immunoreactivity in tumor tissues was increased in recurrence group after TAM therapy compared to non-recurrence group after TAM therapy. Phosphorylation of Akt, extracellular signal-regulated kinase (ERK), and p38 kinase were all increased in TAMR-MCF-7 cells. Inhibition of phosphoinositide 3-kinase (PI3K) suppressed the transactivation of the aromatase gene and its enzyme activity. Furthermore, we have also shown that PI3K/Akt-dependent cAMP-response element binding protein (CREB) activation was required for the enhanced expression of aromatase in TAMR-MCF-7 cells. Our findings suggest that aromatase expression is up-regulated in TAM-resistant breast cancer via PI3K/Akt-dependent CREB activation.

Keywords: TAMR-MCF-7, CREB, estrogen receptor, aromatase

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Authors: Ashok K. Gupta

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Over the past few decades, since the bio-engineering revolution, autologous cell therapy (ACT) has become a rapidly evolving field. Currently, this form of therapy has broad applications in modern medicine and plastic surgery, ranging from the treatment/improvement of wound healing to life-saving operations. A study was conducted on 50 patients having to disfigure, and deform post burn scars and was treated by injection of extracted, refined adipose tissue grafts with their unique stem cell properties. To compare the outcome, a control of 20 such patients was treated with conventional skin or soft-tissue flaps or skin grafting, and a control of 10 was treated with more advanced microsurgical techniques such as Pre-fabricated flaps/pre laminated flaps / free flaps. Assessment of fat volume and survival post- follow up period was done by radiological aid, using MRI and clinically (Survival of the autograft and objective parameters for scar elasticity were evaluated skin elasticity parameters 3 to 9 months postoperatively). Recently, an enzyme that is involved in collagen crosslinking in fibrotic tissue, lysyl hydroxylase (LH2), was identified. This enzyme is normally active in bone and cartilage but hardly in the skin. It has been found that this enzyme is highly expressed in scar tissue and subcutaneous fat; this is in contrast to the dermis, where the enzyme is hardly expressed. Adipose tissue-derived stem cell injections are an effective method in the treatment of various extensive post-burn scar deformities that makes it possible to re-create the lost sub-dermal tissue for improvement in the function of involved joint movements.

Keywords: adipose tissue-derived stem cell injections, treatment of various extensive post-burn scar deformities, re-create the lost sub-dermal tissue, improvement in function of involved joint movements

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Authors: J. Hemanth, B. G. Shivaprakash, S. V. Dinesh

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The locally available Lateritic soil in Dakshina Kanadda and Udupi districts are traditionally being used as building blocks for construction purpose but they do not meet the conventional requirements (L L ≤ 25% & P I ≤6%) and desired four days soaked CBR value to be used as a sub-base course material in pavements. In order to improve its properties to satisfy the Atterberg’s Limits, the soil is blended with sand, cement and quarry dust at various percentages and also to meet the CBR strength requirements, individual and combined gradation of various sized aggregates along with Laterite soil and other filler materials has been done for coarse graded granular sub-base materials (Grading II and Grading III). The effect of additives blended with lateritic soil and aggregates are studied in terms of Atterberg’s limits, compaction, California Bearing Ratio (CBR), and permeability. It has been observed that the addition of sand, cement and quarry dust are found to be effective in improving Atterberg’s limits, CBR values, and permeability values. The obtained CBR and permeability values of Grading III, and Grading II materials found to be sufficient to be used as sub-base course for low volume roads and high volume roads respectively.

Keywords: lateritic soil, sand, quarry dust, gradation, sub-base course, permeability

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Authors: Ahmet Kurklu, Abdussamed Sarp, Gokmen Arikan, Akin Eren, Arif Ulu, Ferit Cakir

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Our natural resources are diminishing day by day with the needs of the growing world population. There is a danger that these resources will be depleted if they are not used in a controlled manner. As a result of the rapid increase in the consumption of limited natural resources, one of the issues where studies have gained importance is recycling. Many countries have carried out various research and development activities on recycling and reuse to prevent wastage of resources. For sustainable and healthy living, the limited amount of raw material resources in nature should be consumed consciously, and the necessary awareness should be given for recycling activities. One of the sectors where the consumption of raw materials is high is the construction sector. With the changing consumption habits of the evolving technology in the construction sector, the need to use special concrete along with the normal concrete has arisen. With the increasing need for specialty concretes, polymer concrete, which was discovered in the early 1900s, has evolved to the present day. Polymer concretes are special concretes with high strength, water impermeability, resistance to chemical action, and low surface roughness. Thanks to these properties, they find wide applications in many fields such as swimming pools, drainage systems, repair works. In the study, the effect of using recycled aggregates instead of natural aggregates in the production of polymer concrete on the performance of polymer concrete is investigated. In the experiments conducted for this purpose, the use of natural aggregate is reduced at certain rates, and instead, recycled aggregate is added at the same rate. The recycled aggregate to be used in the study is obtained from the polymer concrete drainage channel production facility of Mert Casting Co., Istanbul, Turkey. In order to clearly observe the effect of recycled materials on the product in the study, the other components (resin, hardener, accelerator, and additive) are kept constant in the concrete mix. In the study, fresh and hardened concrete tests are to be carried out on the mixes to be prepared.

Keywords: concrete, mechanical properties, polymer concrete, recycle aggregate

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Authors: Md. Z. Alam, Amal A. Elgharbawy, Muhammad Moniruzzaman, Nassereldeen A. Kabbashi, Parveen Jamal

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The enzymatic hydrolysis of lignocellulosic biomass is one of the obstacles in the process of sugar production, due to the presence of lignin that protects the cellulose molecules against cellulases. Although the pretreatment of lignocellulose in ionic liquid (IL) system has been receiving a lot of interest; however, it requires IL removal with an anti-solvent in order to proceed with the enzymatic hydrolysis. At this point, introducing a compatible cellulase enzyme seems more efficient in this process. A cellulase enzyme that was produced by Trichoderma reesei on palm kernel cake (PKC) exhibited a promising stability in several ILs. The enzyme called PKC-Cel was tested for its optimum pH and temperature as well as its molecular weight. One among evaluated ILs, 1,3-diethylimidazolium dimethyl phosphate [DEMIM] DMP was applied in this study. Evaluation of six factors was executed in Stat-Ease Design Expert V.9, definitive screening design, which are IL/ buffer ratio, temperature, hydrolysis retention time, biomass loading, cellulase loading and empty fruit bunches (EFB) particle size. According to the obtained data, IL-enzyme system shows the highest sugar concentration at 70 °C, 27 hours, 10% IL-buffer, 35% biomass loading, 60 Units/g cellulase and 200 μm particle size. As concluded from the obtained data, not only the PKC-Cel was stable in the presence of the IL, also it was actually stable at a higher temperature than its optimum one. The reducing sugar obtained was 53.468±4.58 g/L which was equivalent to 0.3055 g reducing sugar/g EFB. This approach opens an insight for more studies in order to understand the actual effect of ILs on cellulases and their interactions in the aqueous system. It could also benefit in an efficient production of bioethanol from lignocellulosic biomass.

Keywords: cellulase, hydrolysis, lignocellulose, pretreatment

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Authors: Frieda Eivazi, Nikita L. Mullings

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Soils are recipient for surfactants in herbicide formulations. Surfactants entering the soil environment can possibly disrupt different chemical, physical and biological interactions. Therefore, it is critical that we understand the fate, behavior and transport of surfactants upon entering the soil. A comprehensive study was conducted to examine effect of surfactants on nutrient uptake, microbial community, and enzyme activity. The research was conducted in the greenhouse growing corn (Zea mays) as a test plant in a factorial experiment (three surfactants at two different rates with control, and three herbicides) organized as randomized blocked design. Surfactants evaluated were Activator 90, Agri-Dex, and Thrust; herbicides were glyphosate, atrazine, and bentazon. Treatments examined were surfactant only, herbicide only, and surfactant + herbicide combinations. Corn was planted in fertilized soils (silt loam and silty clay) with moisture content maintained at the field capacity for optimum growth. This paper will report results of above mentioned treatments on acid phosphatase, beta-glucosidase, arylsulfatase, beta-glucosaminidase, and dehydrogenase activities. In general, there were variations in the enzyme activities with some inhibition and some being enhanced by the treatments. Activator 90 appeared to have the highest inhibitory effect on enzymatic activities. Atrazine application significantly decreased the activities of acid phosphatase, beta-glucosidase, and dehydrogenase in both soils; however, combination of Atrazine + Agridex increased the acid phosphatase activity while significantly inhibiting the other enzyme activities in soils. It was concluded that long-term field studies are needed to validate changes in nutrient uptake, microbial community and enzyme activities due to surfactant-herbicide combination effects.

Keywords: herbicides, nutrient cycling, soil enzymes, surfactant

Procedia PDF Downloads 219

Authors: Mallika Supa-Aksorn, Buaream Maneewan, Jiraporn Rojtinnakorn

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For animals, trypsin and chymotrypsin are the 2 proteases that play the important role in protein digestion and involving in growth rate. In many animals, these two enzymes are indicated as growth parameter by feed. Although enzyme assay at optimal condition is significant for its accuracy activity determination. There is less report of trypsin and chymotrypsin. Therefore, in this study, optimization of pH and temperature for trypsin (T) and chymotrypsin (C) in economic species; i.e. Nile tilapia (Oreochromis niloticus), sand goby (Oxyeleotoris marmoratus), giant freshwater prawn (Macrobachium rosenberchii) and native chicken (Gallus gallus) were investigated. Each enzyme of each species was assaying for its specific activity with variation of pH in range of 2-12 and temperature in range of 30-80 °C. It revealed that, for Nile tilapia, T had optimal condition at pH 9 and temperature 50-80 °C, whereas C had optimal condition at pH 8 and temperature 60 °C. For sand goby, T had optimal condition at pH 7 and temperature of 50 °C, while C had optimal condition at pH 11 and temperature of 70-75 °C. For juvenile freshwater prawn, T had optimal condition at pH 10-11 and temperature of 60-65 °C, C had optimal condition at pH 8 and temperature of 70°C. For starter native chicken, T has optimal condition at pH 7 and temperature of 70 °C, whereas C had o optimal condition at pH 8 and temperature of 60°C. This information of optimal conditions will be high valuable in further for, actual enzyme measurement of T and C activities that benefit for growth and feed analysis.

Keywords: trypsin, chymotrypsin, Oreochromis niloticus, Oxyeleotoris marmoratus, Macrobachium rosenberchii, Gallus gallus

Procedia PDF Downloads 233

Authors: Charles Kwasi Antwi, Mohammad Naushad Emmambux, Natalia Rosa-Sibakov

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The consumption of high-fibre snacks is beneficial in reducing the prevalence of most non-communicable diseases and improving human health. However, using high-fibre flour to produce snacks by extrusion cooking reduces the expansion ratio of snacks, thereby decreasing sensory properties and consumer acceptability of the snack. The study determines the effects of adding Viscozyme®-treated sorghum bran on the properties of extruded sorghum snacks with the aim of producing high-fibre expanded snacks with acceptable quality. With a twin-screw extruder, sorghum endosperm flour [by decortication] with and without sorghum bran and with enzyme-treated sorghum bran was extruded at high shear rates with feed moisture of 20%, feed rate of 10 kg/hr, screw speed of 500 rpm, and temperature zones of 60°C, 70°C, 80°C, 140°C, and 140°C toward the die. The expanded snacks that resulted from this process were analysed in terms of their physical (expansion ratio, bulk density, colour profile), chemical (soluble and insoluble dietary fibre), and functional (water solubility index (WSI) and water absorption index (WAI)) characteristics. The expanded snacks produced from refined sorghum flour enriched with Viscozyme-treated bran had similar expansion ratios to refined sorghum flour extrudates, which were higher than those for untreated bran-sorghum extrudate. Sorghum extrudates without bran showed higher values of expansion ratio and low values of bulk density compared to the untreated bran extrudates. The enzyme-treated fibre increased the expansion ratio significantly with low bulk density values compared to untreated bran. Compared to untreated bran extrudates, WSI values in enzyme-treated samples increased, while WAI values decreased. Enzyme treatment of bran reduced particle size and increased soluble dietary fibre to increase expansion. Lower particle size suggests less interference with bubble formation at the die. Viscozyme-treated bran-sorghum composite flour could be used as raw material to produce high-fibre expanded snacks with improved physicochemical and functional properties.

Keywords: extrusion, sorghum bran, decortication, expanded snacks

Procedia PDF Downloads 52

Authors: James Britton, Vijaya Krishna, Manus Biggs, Abhay Pandit

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Regeneration of the spinal cord after injury remains a great challenge due to the complexity of this organ. Inflammation and gliosis at the injury site hinder the outgrowth of axons and hence prevent synaptic reconnection and reinnervation. Hyaluronic acid (HA) is the main component of the spinal cord extracellular matrix and plays a vital role in cell proliferation and axonal guidance. In this study, we have synthesized and characterized a photo-cross-linkable HA-tyramine (tyr) hydrogel from a chemical, mechanical, electrical, biological and structural perspective. From our experimentation, we have found that HA-tyr can be synthesized with controllable degrees of tyramine substitution using click chemistry. The complex modulus (G*) of HA-tyr can be tuned to mimic the mechanical properties of the native spinal cord via optimization of the photo-initiator concentration and UV exposure. We have examined the degree of tyramine-tyramine covalent bonding (polymerization) as a function of UV exposure and photo-initiator use via Photo and Nuclear magnetic resonance spectroscopy. Both swelling and enzymatic degradation assays were conducted to examine the resilience of our 3D printed hydrogel constructs in-vitro. Using a femtosecond 780nm laser, the two-photon polymerization of HA-tyr hydrogel in the presence of riboflavin photoinitiator was optimized. A laser power of 50mW and scan speed of 30,000 μm/s produced high-resolution spatial patterning within the hydrogel with sustained mechanical integrity. Using dorsal root ganglion explants, the cytocompatibility of photo-crosslinked HA-tyr was assessed. Using potentiometry, the electrical conductivity of photo-crosslinked HA-tyr was assessed and compared to that of native spinal cord tissue as a function of frequency. In conclusion, we have developed a biocompatible hydrogel that can be used for photolithographic 3D printing to fabricate tissue engineered constructs for neural tissue regeneration applications.

Keywords: 3D printing, hyaluronic acid, photolithography, spinal cord injury

Procedia PDF Downloads 131

Authors: Wen-Shyong Tzou, Chih-Ching Huang, Chin-Hwa Hu, Ying-Tsang Lo, Tun-Wen Pai, Chia-Yin Chiang, Chung-Hao Li, Hong-Jyuan Jian

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Plasmin plays an important role in the human circulatory system owing to its catalytic ability of fibrinolysis. The immediate injection of plasmin in patients of strokes has intrigued many scientists to design vectors that can transport plasmin to the desired location in human body. Here we predict the structure of human plasmin and investigate the interaction of plasmin with the gold-nanoparticle. Because the crystal structure of plasminogen has been solved, we deleted N-terminal domain (Pan-apple domain) of plasminogen and generate a mimic of the active form of this enzyme (plasmin). We conducted a simulated annealing process on plasmin and discovered a very large conformation occurs. Kringle domains 1, 4 and 5 had been observed to leave its original location relative to the main body of the enzyme and the original doughnut shape of this enzyme has been transformed to a V-shaped by opening its two arms. This observation of conformational change is consistent with the experimental results of neutron scattering and centrifugation. We subsequently docked the plasmin on the simulated gold surface to predict their interaction. The V-shaped plasmin could utilize its Kringle domain and catalytic domain to contact the gold surface. Our findings not only reveal the flexibility of plasmin structure but also provide a guide for the design of a plasmin-gold nanoparticle.

Keywords: docking, gold nanoparticle, molecular simulation, plasmin

Procedia PDF Downloads 452

Authors: M. Umar Dahot, G. S. Mangrio

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In this study Agro-industrial waste was used as a carbon source, which is a low cost substrate. Along with this, various sugars and molasses of 2.5% and 5% were investigated as substrate/carbon source for the growth of A.niger and Pectinase production. Different nitrogen sources were also used. An overview of results obtained show that 5% sucrose, 5% molasses and 0.4% (NH4)2SO4 were found the best carbon and nitrogen sources for the production of pectinase by A. niger. The maximum production of pectinase (26.87units/ml) was observed at pH 6.0 after 72 hrs incubation. The optimum temperature for the maximum production of pectinase was achieved at 35ºC when maximum production of pectinase was obtained as 28.25Units/ml.Pectinase enzyme was purified with ammonium sulphate precipitation and dialyzed sample was finally applied on gel filtration chromatography (Sephadex G-100) and Ion Exchange DEAE A-50. The enzyme was purified 2.5 fold by gel chromatography on Sephadex G-100 and Four fractions were obtained, Fraction 1, 2, 4 showed single band while Fraction -3 showed multiple bands on SDS Page electrophoresis. Fraction -3 was pooled, dialyzed and separated on Sephdex A-50 and two fractions 3a and 3b showed single band. The molecular weights of the purified fractions were detected in the range of 33000 ± 2000 and 38000± 2000 Daltons. The purified enzyme was specifically most active with pure pectin, while pectin, Lemon pectin and orange peel given lower activity as compared to (control). The optimum pH and temperature for pectinase activity was found between pH 5.0 and 6.0 and 40°- 50°C, respectively. The enzyme was stable over the pH range 3.0-8.0. The thermostability of was determined and it was observed that the pectinase activity is heat stable and retains activity more than 40% when incubated at 90°C for 10 minutes. The pectinase activity of F3a and F3b was increased with different metal ions. The Pectinase activity was stimulated in the presence of CaCl2 up to 10-30%. ZnSO4, MnSO4 and Mg SO4 showed higher activity in fractions F3a and F3b, which indicates that the pectinase belongs to metalo-enzymes. It is concluded that A. niger is capable to produce pH stable and thermostable pectinase, which can be used for industrial purposes.

Keywords: pectinase, a. niger, production, purification, characterization

Procedia PDF Downloads 387

Authors: Evgeniya Y. Yuzbasheva, Tigran V. Yuzbashev, Natalia I. Perkovskaya, Elizaveta B. Mostova

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Cell-surface display of lipase is of great interest as it has many applications in the field of biotechnology owing to its unique advantages: simplified product purification, and cost-effective downstream processing. One promising area of application for whole-cell biocatalysts with surface displayed lipase is biodiesel synthesis. Biodiesel is biodegradable, renewable, and nontoxic alternative fuel for diesel engines. Although the alkaline catalysis method has been widely used for biodiesel production, it has a number of limitations, such as rigorous feedstock specifications, complicated downstream processes, including removal of inorganic salts from the product, recovery of the salt-containing by-product glycerol, and treatment of alkaline wastewater. Enzymatic synthesis of biodiesel can overcome these drawbacks. In this study, Lip2p lipase was displayed on Yarrowia lipolytica cells via C- and N-terminal fusion variant. The active site of lipase is located near the C-terminus, therefore to prevent the activity loosing the insertion of glycine-serine linker between Lip2p and C-domains was performed. The hydrolytic activity of the displayed lipase reached 12,000–18,000 U/g of dry weight. However, leakage of enzyme from the cell wall was observed. In case of C-terminal fusion variant, the leakage was occurred due to the proteolytic cleavage within the linker peptide. In case of N-terminal fusion variant, the leaking enzyme was presented as three proteins, one of which corresponded to the whole hybrid protein. The calculated number of recombinant enzyme displayed on the cell surface is approximately 6–9 × 105 molecules per cell, which is close to the theoretical maximum (2 × 106 molecules/cell). Thus, we attribute the enzyme leakage to the limited space available on the cell surface. Nevertheless, cell-bound lipase exhibited greater stability to short-term and long-term temperature treatment than the native enzyme. It retained 74% of original activity at 60°C for 5 min of incubation, and 83% of original activity after incubation at 50°C during 5 h. Cell-bound lipase had also higher stability in organic solvents and detergents. The developed whole-cell biocatalyst was used for recycling biodiesel synthesis. Two repeated cycles of methanolysis yielded 84.1–% and 71.0–% methyl esters after 33–h and 45–h reactions, respectively.

Keywords: biodiesel, cell-surface display, lipase, whole-cell biocatalyst

Procedia PDF Downloads 461

Authors: Zhanara Suleimenova, Raushan Blieva, Aigerim Zhakipbekova, Inkar Tapenbayeva, Zhanar Narmuratova

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Green processes are based on innovative technologies that do not negatively affect the environment. Industrial enzymes originated from biological systems can effectively contribute to sustainable development through being isolated from microorganisms which are fermented using primarily renewable resources. Many widespread microorganisms secrete a significant amount of biocatalysts into the environment, which greatly facilitates the task of their isolation and purification. The ability to control the enzyme production through the regulation of their biosynthesis and the selection of nutrient media and cultivation conditions allows not only to increase the yield of enzymes but also to obtain enzymes with certain properties. In this regard, large potentialities are embedded in immobilized cells. Enzyme production technology in a secreted active form enabling industrial application on an economically feasible scale has been developed. This method is based on the immobilization of enzyme producers on a solid career. Immobilizing has a range of advantages: decreasing the price of the final product, absence of foreign substances, controlled process of enzyme-genesis, the ability of various enzymes' simultaneous production, etc. Design of proposed equipment gives the opportunity to increase the activity of immobilized cell culture filtrate comparing to free cells, growing in periodic culture conditions. Such technology allows giving a 10-times raise in culture productivity, to prolong the process of fungi cultivation and periods of active culture liquid generation. Also, it gives the way to improve the quality of filtrates (to make them more clear) and exclude time-consuming processes of recharging fermentative vials, that require manual removing of mycelium.

Keywords: industrial enzymes, immobilization, submerged fermentation, microscopic fungi

Procedia PDF Downloads 116

Authors: Palwinder Singh

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Beyond the conventional mode of working with anti-inflammatory agents through enzyme inhibition, herein, an alternate substrate of cyclooxygenase-2 was developed. Proline centered pentapeptide iso-conformational to arachidonic acid exhibited appreciable selectivity for COX-2 overcoming acetic acid and formalin induced pain in rats to almost 80% and was treated as a substrate by the enzyme. Remarkably, COX-2 metabolized the pentapeptide into small fragments consisting mainly of di- and tri-peptides that ensured the safe breakdown of the peptide under in-vivo conditions. The kinetic parameter Kcat/Km for COX-2 mediated metabolism of peptide 6.3 x 105 M-1 s-1 was quite similar to 9.5 x 105 M-1 s-1 for arachidonic acid. Evidenced by the dynamic molecular studies and the use of Y385F COX-2, it was observed that the breakage of the pentapeptide has probably taken place through H-bond activation of the peptide bond by the side chains of Y385 and S530.

Keywords: small peptides, anti-inflammatory agents, cyclooxygenase-2, unnatural substrates

Procedia PDF Downloads 48

Authors: Eleftheria Barouni, Theano Petsi, Argyro Bekatorou, Dionysos Kolliopoulos, Dimitrios Vasileiou, Panayiotis Panas, Maria Kanellaki, Athanasios A. Koutinas

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The aim of the present work was the production and use of a composite filter (TC/starch), containing rennin enzyme, in continuous system and in successive fermentation batches (SFB) for milk coagulation in order to compare the operational stability of both systems and cheese production cost. Tubular cellulose (TC) was produced after removal of lignin from lignocellulosic biomass using several procedures, e.g. alkaline treatment [1] and starch gel was added for the reduction of TC tubes dimensions to micro- and nano- range[2]. Four immobilized biocatalysts were prepared using different ways of the enzyme entrapment. 1) TC/ rennin (rennin entrapped in the tubes of TC), 2) TC/SG-rennin (rennin entrapped in the tubes of the composite), 3) TC-SG/rennin (rennin entrapped into the layer of starch gel) and 4) TC/rennin- SG/rennin (rennin is entrapped both in the tubes of the TC and into the layer of starch gel). Firstly these immobilized biocatalysts were examined in ten SFB regarding the coagulation time and their activity All the above immobilized biocatalysts remained active and the coagulation time was ranged from 90 to 480, 120-480, 330-510, and 270-540 min for (1), (2), (3), and (4) respectively. The quality of the cheese was examined through the determination of volatile compounds by SPME GC/MS analysis. These results encouraged us to study a continuous coagulation system of milk. Even though the (1) immobilized biocatalyst gave lower coagulation time, we used the (2) immobilized biocatalyst in the continuous system. The results were promising.

Keywords: tubular cellulose, starch gel, composite biocatalyst, Rennin, milk coagulation

Procedia PDF Downloads 293

Authors: Chayanon Boonyuid

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This paper experiments the effects of low-density polyethylene (LDPE) on the structural strength and permeability of hot-mix-asphalt (HMA) pavements. Different proportions of bitumen (4%, 4.5%, 5%, 5.5% and 6% of total aggregates) and plastic (5%, 10% and 15% of bitumen) contents in HMA mixtures were investigated to estimate the optimum mixture of bitumen and plastic in HMA pavement with long-term performance. Marshall Tests and Falling Head Tests were performed to experiment the structure strength and permeability of HMA mixtures with different percentages of plastic materials and bitumen. The laboratory results show that the optimum binder content was 5.5% by weight of aggregates with higher contents of plastic materials, increase structural stability, reduce permanent deformation, increase ductility, and improve fatigue life of HMA pavements. The use of recycled plastic shopping bags can reduce the use of bitumen content by 0.5% - 1% in HMA mixtures resulting in cheaper material costs with better long-term performance. The plastic materials increase the impermeability of HMA pavements. This study has two-fold contributions: optimum contents of both bitumen and plastic materials in HMA mixtures and the impacts of plastic materials on the permeability of HMA pavements.

Keywords: plastic bags, bitumen, structural strength, permeability

Procedia PDF Downloads 123