Search results for: immobilized metal affinity chromatography (IMAC)

Authors: Duygu Çimen, Nilay Bereli, Adil Denizli

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In this study is to synthesis magnetic nanoparticles for purify protein C. For this aim, N-Methacryloyl-(L)-histidine methyl ester (MAH) containing 2-hydroxyethyl methacrylate (HEMA) based magnetic nanoparticles were synthesized by using micro-emulsion polymerization technique for templating protein C via metal chelation. The obtained nanoparticles were characterized with Fourier transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM), zeta-size analysis and electron spin resonance (ESR) spectroscopy. After that, they were used for protein C purification from aqueous solution to evaluate/optimize the adsorption condition. Hereby, the effecting factors such as concentration, pH, ionic strength, temperature, and reusability were evaluated. As the last step, protein C was determined with sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Keywords: immobilized metal affinity chromatography (IMAC), magnetic nanoparticle, protein C, hydroxyethyl methacrylate (HEMA)

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Authors: Naureen Akhtar

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The stability of the protein in detergent-containing solution is the key for its successful crystallisation. Fluorescence-detection size-exclusion chromatography (FSEC) is a potential approach for screening monodispersity as well as the stability of protein in a detergent-containing-solution. In this present study, covalently linked Green Fluorescent Protein (GFP) to bacterial nitrate transporter, NarU from Escherichia coli was studied for pre-crystallisation trials by FSEC. Immobilised metal ion affinity chromatography (IMAC) and gel filtration were employed for their purification. The main objectives of this study were over-expression, detergent screening and crystallisation of nitrate transporter proteins. This study could not produce enough proteins that could realistically be taken forward to achieve the objectives set for this particular research. In future work, different combinations of variables like vectors, tags, creation of mutant proteins, host cells, position of GFP (N- or C-terminal) and/or membrane proteins would be tried to determine the best combination as the principle of technique is still promising.

Keywords: transporters, detergents, over-expression, crystallography

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Authors: K. Verma, v. S. Moholkar

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In developed countries, water pollution caused by industrial discharge has emerged as a significant environmental concern over the past decades. However, despite ongoing efforts, a fully effective and sustainable remediation strategy has yet to be identified. This paper describes how enzymatic and sonochemical treatments have demonstrated great promise in degrading bio-refractory pollutants. Mainly, a compelling area of interest lies in the combined technique of sono-enzymatic treatment, which has exhibited a synergistic enhancement effect surpassing that of the individual techniques. This study employed the covalent attachment method to immobilize Laccase from Trametes versicolor onto amino-functionalized magnetic Fe₃O₄ nanoparticles. To comprehensively characterize the synthesized free nanoparticles and the laccase-immobilized nanoparticles, various techniques such as X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), scanning electron microscope (SEM), vibrating sample magnetometer (VSM), and surface area through Brunauer-Emmett-Teller (BET) were employed. The size of immobilized Fe₃O₄@Laccase was found to be 60 nm, and the maximum loading of laccase was found to be 24 mg/g of nanoparticle. An investigation was conducted to study the effect of various process parameters, such as immobilized Fe₃O₄ Laccase dose, temperature, and pH, on the % Chemical oxygen demand (COD) removal as a response. The statistical design pinpointed the optimum conditions (immobilized Fe₃O₄ Laccase dose = 1.46 g/L, pH = 4.5, and temperature = 66 oC), resulting in a remarkable 65.58% COD removal within 60 minutes. An even more significant improvement (90.31% COD removal) was achieved with ultrasound-assisted enzymatic reaction utilizing a 10% duty cycle. The investigation of various kinetic models for free and immobilized laccase, such as the Haldane, Yano, and Koga, and Michaelis-Menten, showed that ultrasound application impacted the kinetic parameters Vmax and Km. Specifically, Vmax values for free and immobilized laccase were found to be 0.021 mg/L min and 0.045 mg/L min, respectively, while Km values were 147.2 mg/L for free laccase and 136.46 mg/L for immobilized laccase. The lower Km and higher Vmax for immobilized laccase indicate its enhanced affinity towards the substrate, likely due to ultrasound-induced alterations in the enzyme's confirmation and increased exposure of active sites, leading to more efficient degradation. Furthermore, the toxicity and Liquid chromatography-mass spectrometry (LC-MS) analysis revealed that after the treatment process, the wastewater exhibited 70% less toxicity than before treatment, with over 25 compounds degrading by more than 75%. At last, the prepared immobilized laccase had excellent recyclability retaining 70% activity up to 6 consecutive cycles. A straightforward manufacturing strategy and outstanding performance make the recyclable magnetic immobilized Laccase (Fe₃O₄ Laccase) an up-and-coming option for various environmental applications, particularly in water pollution control and treatment.

Keywords: kinetic, laccase enzyme, sonoenzymatic, ultrasound irradiation

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Authors: Satyendra P. Chaurasia, Aditi Sharma, Ajay K. Dalai

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The esterification of Docosahexaenoic acid (DHA) with glycerol using immobilized Mucor mie-hei lipase (MML) and Rhizopus oryzae lipase (ROL) have been studied in the present paper to synthesize triglycerides (TG) rich in DHA. Both immobilized lipases (MML and ROL), and their support materials (immobead-150 and ion-exchange resin) were characterized and compared for surface properties with BET, for chemical functional groups with FT-IR, and for particle size distribution with particle size analyzer. The most suitable reaction conditions for synthesis of DHA rich TG in biphasic solvent system were found as 1:3 (wt/wt) glycerol to DHA ratio, 1:1 (wt/wt) buffer to DHA ratio, 1:1 (wt/wt) solvent to DHA ratio at 50 ºC temperature, and 600 rpm speed of agitation with 100 mg of immobilized lipases. Maximum 95.9 % esterification was obtained with immobilized MML in 14 days reaction with formation of 65.7 wt% DHA rich TG. Whereas, immobilized ROL has shown formation of only 23.8 wt% DHA rich TG with total 78.9 % esterification in 15 days. Additionally, repeated use of both immobilized lipases was con-ducted up to five cycles, indicated 50.4% and 41.2 % activity retention after fifth repeated use of immobilized MML and ROL, respectively.

Keywords: DHA, immobilized Mucor miehei lipase, Rhizopus oryzae lipase, esterification

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Authors: Eleni Poulou-Sidiropoulou, Charalampos N. Bompas, Martina Samiotaki, Alexios Vlamis-Gardikas

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The glutaredoxin (Grx) and thioredoxin (Trx) systems keep the intracellular environment reduced in almost all organisms. In Escherichia coli (E. coli), the Grx system relies on NADPH+ to reduce GSH reductase (GR), the latter reducing oxidized diglutathione to glutathione (GSH) which in turn reduces cytosolic Grxs, the electron donors for different intracellular substrates. In the Trx system, GR and GSH are replaced by Trx reductase (TrxR). Three of the Grxs of E. coli (Grx1, 2, 3) are reduced by GSH, while Grx4 is likely reduced by TrxR. Trx1 and Grx1 from E. coli may reduce ribonucleotide reductase Ia to ensure a constant supply of deoxyribonucleotides for the synthesis of DNA. The role of the other three Grxs is relatively unknown, especially for Grx2 that may amount up to 1 % of total cellular protein in the stationary phase of growth. The protein is known as a potent antioxidant, but no specific functions have been attributed to it. Herein, affinity chromatography of cellular extracts on immobilized Grx2, followed by MS analysis of the resulting eluates, was employed to identify protein ligands that could provide insights into the biological role of Grx2. Ionic, strong non-covalent, and covalent (disulfide) interactions with relevant proteins were detected. As a means of verification, the identified ligands were subjected to in silico docking with monothiol Grx2. In other experiments, protein extracts from E. coli cells lacking the gene for Grx2 (grxB) were compared to those of wild type. Taken together, the two approaches suggest that Grx2 is involved in protein synthesis, nucleotide metabolism, DNA damage repair, stress responses, and various metabolic processes. Grx2 appears as a versatile protein that may participate in a wide range of biological pathways beyond its known general antioxidant function.

Keywords: Escherichia coli, glutaredoxin 2, interactome, thiol-disulfide oxidoreductase

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Authors: Chang, Ya-Ju, Hsieh, Pei-Hsin, Wu, Jui-Chuang, Chen-Yang, Yui Whei

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A mesoporous silica material was prepared to apply to the lateral-flow immunochromatography for detecting a model biosample. The probe antibody is immobilized on the silica surface as the test line to capture its affinity antigen, which laterally flows through the chromatography strips. The antigen is labeled with nano-gold particles, such that the detection can be visually read out from the test line without instrument aids. The result reveals that the mesoporous material provides a vast area for immobilizing the detection probes. Biosening surfaces corresponding with a positive proportion of detection signals is obtained with the biosample loading.

Keywords: mesoporous silica, immunochromatography, lateral-flow strips, biosensors, nano-gold particles

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Authors: K.Narasimhulu, Y. Pydi Setty

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The objective of this present study is the optimization of process parameters in biosorption of Ni(II) and Cd(II) ions by Pseudomonas putida using Response Surface Methodology in a Packed bed bioreactor. The experimental data were also tested with theoretical models to find the best fit model. The present paper elucidates RSM as an efficient approach for predictive model building and optimization of Ni(II) and Cd(II) ions using Pseudomonas putida. In packed bed biosorption studies, comparison of the breakthrough curves of Ni(II) and Cd(II) for Agar immobilized and PAA immobilized Pseudomonas putida at optimum conditions of flow rate of 300 mL/h, initial metal ion concentration of 100 mg/L and bed height of 20 cm with weight of biosorbent of 12 g, it was found that the Agar immobilized Pseudomonas putida showed maximum percent biosorption and bed saturation occurred at 20 minutes. Optimization results of Ni(II) and Cd(II) by Pseudomonas putida from the Design Expert software were obtained as bed height of 19.93 cm, initial metal ion concentration of 103.85 mg/L, and flow rate of 310.57 mL/h. The percent biosorption of Ni(II) and Cd(II) is 87.2% and 88.2% respectively. The predicted optimized parameters are in agreement with the experimental results.

Keywords: packed bed bioreactor, response surface mthodology, pseudomonas putida, biosorption, waste water

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Authors: Oktira Roka Aji, Maelita R. Moeis, Ihsanawati, Ernawati A. Giri-Rachman

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Globally, tuberculosis (TB) remains a leading cause of death. The emergence of multidrug-resistant strains and extensively drug-resistant strains have become a major public concern. One of the potential candidates for drug target is the cytoplasmic domain of PhoR Histidine Kinase, a part of the Two Component System (TCS) PhoR-PhoP in Mycobacterium tuberculosis (Mtb). TCS PhoR-PhoP relay extracellular signal to control the expression of 114 virulent associated genes in Mtb. The 3D structure of PhoR cytoplasmic domain is needed to screen novel drugs using structure based drug discovery. The PhoR cytoplasmic domain from Mtb H37Rv was overexpressed in E. coli BL21(DE3), then purified using IMAC Ni-NTA Agarose his-tag affinity column and DEAE-ion exchange column chromatography. The molecular weight of the purified protein was estimated to be 37 kDa after SDS-PAGE analysis. This sample was used for pre-crystallization screening by applying sitting drop vapor diffusion method using Natrix (HR2-116) 48 solutions crystal screen kit at 25ºC. Needle-like crystals were observed after the seventh day of incubation in test solution No.47 (0.1 M KCl, 0.01 M MgCl2.6H2O, 0.05 M Tris-Cl pH 8.5, 30% v/v PEG 4000). Further testing is required for confirming the crystal.

Keywords: tuberculosis, two component system, histidine kinase, needle-like crystals

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Authors: Leila Mosafa, Majid Moghadam, Mohammad Shahedi

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In this work, pectinase was immobilized on the surface of silica-coated magnetite nanoparticles via covalent attachment. The magnetite-immobilized enzyme was characterized by Fourier transform infrared spectroscopy, X-ray powder diffraction, scanning electron microscopy and vibrating sample magnetometry techniques. Response surface methodology using Minitab Software was applied for statistical designing of operating conditions in order to immobilize pectinase on magnetic nanoparticles. The optimal conditions were obtained at 30°C and pH 5.5 with 42.97 µl pectinase for 2 h. The immobilization yield was 50.6% at optimized conditions. Compared to the free pectinase, the immobilized pectinase was found to exhibit enhanced enzyme activity, better tolerance to the variation of pH and temperature, and improved storage stability. Both free and immobilized samples reduced the viscosity of apple juice from 1.12 to 0.88 and 0.92 mm2s-1, respectively, after 30 min at their optimum temperature. Furthermore, the immobilized enzyme could be reused six consecutive cycles and the efficiency loss in viscosity reduction was found to be only 8.16%.

Keywords: magnetite nanoparticles, pectinase enzyme, immobilization, juice clarification, enzyme activity

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Authors: Shanna Swart, Caryn Fenner, Athanasios Kotsiopoulos, Susan Harrison

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Linear alkanes are produced as by-products from the increasing use of gas-to-liquid fuel technologies for synthetic fuel production and offer great potential for value addition. Their current use as low-value fuels and solvents do not maximize this potential. Therefore, attention has been drawn towards direct activation of these aliphatic alkanes to more useful products such as alcohols, aldehydes, carboxylic acids and derivatives. Cytochrome P450 monooxygenases (P450s) can be used for activation of these aliphatic alkanes using whole-cells or cell-free systems. Some limitations of whole-cell systems include reduced mass transfer, stability and possible side reactions. Since the P450 systems are little studied as cell-free systems, they form the focus of this study. Challenges of a cell-free system include co-factor regeneration, substrate availability and enzyme stability. Enzyme immobilization offers a positive outlook on this dilemma, as it may enhance stability of the enzyme. In the present study, 2 different P450s (CYP153A6 and CYP102A1) as well as the relevant accessory enzymes required for electron transfer (ferredoxin and ferredoxin reductase) and co-factor regeneration (glucose dehydrogenase) have been expressed in E. coli and purified by metal affinity chromatography. Glucose dehydrogenase (GDH), was used as a model enzyme to assess the potential of various enzyme immobilization strategies including; surface attachment on MagReSyn® microspheres with various functionalities and on electrospun nanofibers, using self-assembly based methods forming Cross Linked Enzymes (CLE), Cross Linked Enzyme Aggregates (CLEAs) and spherezymes as well as in a sol gel. The nanofibers were synthesized by electrospinning, which required the building of an electrospinning machine. The nanofiber morphology has been analyzed by SEM and binding will be further verified by FT-IR. Covalent attachment based methods showed limitations where only ferredoxin reductase and GDH retained activity after immobilization which were largely attributed to insufficient electron transfer and inactivation caused by the crosslinkers (60% and 90% relative activity loss for the free enzyme when using 0.5% glutaraldehyde and glutaraldehyde/ethylenediamine (1:1 v/v), respectively). So far, initial experiments with GDH have shown the most potential when immobilized via their His-tag onto the surface of MagReSyn® microspheres functionalized with Ni-NTA. It was found that Crude GDH could be simultaneously purified and immobilized with sufficient activity retention. Immobilized pure and crude GDH could be recycled 9 and 10 times, respectively, with approximately 10% activity remaining. The immobilized GDH was also more stable than the free enzyme after storage for 14 days at 4˚C. This immobilization strategy will also be applied to the P450s and optimized with regards to enzyme loading and immobilization time, as well as characterized and compared with the free enzymes. It is anticipated that the proposed immobilization set-up will offer enhanced enzyme stability (as well as reusability and easy recovery), minimal mass transfer limitation, with continuous co-factor regeneration and minimal enzyme leaching. All of which provide a positive outlook on this robust multi-enzyme system for efficient activation of linear alkanes as well as the potential for immobilization of various multiple enzymes, including multimeric enzymes for different bio-catalytic applications beyond alkane activation.

Keywords: alkane activation, cytochrome P450 monooxygenase, enzyme catalysis, enzyme immobilization

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Authors: Xiao L Dai, Wen X Wei, Shuo Wang, Jia B Li, Yan Wei

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Heavy oil pollution generated from both natural and anthropogenic sources could cause significant damages to the ecological environment, due to the toxicity of some of its constituents. Nowadays, microbial remediation is becoming a promising technology to treat oil pollution owing to its low cost and prevention of secondary pollution; microorganisms are key players in the process. Compared to the free microorganisms, immobilized microorganisms possess several advantages, including high metabolic activity rates, strong resistance to toxic chemicals and natural competition with the indigenous microorganisms, and effective resistance to washing away (in open water system). Many immobilized microorganisms have been successfully used for bioremediation of heavy oil pollution. Considering the broad choices, low cost, simple process, large specific surface area and less impact on microbial activity, modified zeolite were selected as a bio-carrier for bacteria immobilization. Three strains of heavy oil-degrading bacteria Bacillus sp. DL-13, Brevibacillus sp. DL-1 and Acinetobacter sp. DL-34 were immobilized on the modified zeolite under mild conditions, and the bacterial load (bacteria /modified zeolite) was 1.12 mg/g, 1.11 mg/g, and 1.13 mg/g, respectively. SEM results showed that the bacteria mainly adsorbed on the surface or punctured in the void of modified zeolite. The heavy oil degradation efficiency of immobilized bacteria was 62.96%, higher than that of the free bacteria (59.83%). The heavy oil degradation process of immobilized bacteria accords with the first-order reaction equation, and the reaction rate constant is 0.1483 d⁻¹, which was significantly higher than the free bacteria (0.1123 d⁻¹), suggesting that the immobilized bacteria can rapidly start up the heavy oil degradation and has a high activity of heavy oil degradation. The results suggested that immobilized bacteria are promising technology for bioremediation of oil pollution.

Keywords: heavy oil pollution, microbial remediation, modified zeolite, immobilized bacteria

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Authors: Esma Kocaoglu, Oktay Talaz, Huseyin Cavdar, Murat Senturk, Deniz Eki̇nci̇

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Glutathione reductase (GR) is a crucial antioxidant enzyme which is responsible for the maintenance of the antioxidant GSH (glutathione) molecule. Antimalarial effects of some chemical molecules are attributed to their inhibition of GR; thus inhibitors of this enzyme are expected to be promising candidates for the treatment of malaria. In this work, GR inhibitory properties of N-Methylpyrrole derivatives are reported. Firstly, GR was purified by means of affinity chromatography using 2’,5’-ADP-Sepharose 4B as ligand. Enzymatic activity was measured by Beutler’s method. Synthesis of the compounds was approved by thin layer chromatography and column chromatography. Different inhibitor concentrations were used and all compounds were tested in triplicate at each concentration used. It was found that all compounds have better inhibitory activity than the strong GR inhibitor N,N-bis(2-chloroethyl)-N-nitrosourea, especially three molecules, 8m, 8n, and 8q, are the best among them with low micromolar I₅₀ values. Findings of our study indicate that these Schiff base derivatives are strong GR inhibitors which can be used as leads for designation of novel antimalaria candidates.

Keywords: glutathione reductase, antimalaria, inhibitor, enzyme

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Authors: Nikolay E. Dubinin

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Pair-interaction approximations allow to consider a different states of condensed matter from a single position. At the same time, description of an effective pair interaction in transition metal is a hard task since the d-electron contribution to the potential energy in this case is non-pairwise in principle. There are a number of models for transition-metal effective pair potentials. Here we use the Wills-Harrison (WH) approach to calculate pair potentials for Fe, Co, and Ni in crystalline, liquid, and nano states. Last is especially interesting since nano particles of pure transition metals immobilized on the dielectric matrices are widely used in different fields of advanced technologies: as carriers and transmitters of information, as an effective catalytic materials, etc. It is found that the minimum of the pair potential is deeper and oscillations are stronger in nano crystalline state in comparison with the liquid and crystalline states for all metals under consideration.

Keywords: effective pair potential, nanocrystalline state, transition metal, Wills-Harrison approach

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Authors: Li Li, Li-Ru Xia, He-Ye Wang, Xiao-Dong Bi

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In this paper, we presented a highly sensitive immune-affinity monolithic array for detection of α-fetoprotein (AFP) and carcinoembryonic antigen (CEA). Firstly, the epoxy functionalized monolith arrays were fabricated using UV initiated copolymerization method. Scanning electron microscopy (SEM) image showed that the poly(BABEA-co-GMA) monolith exhibited a well-controlled skeletal and well-distributed porous structure. Then, AFP and CEA immune-affinity monolithic arrays were prepared by immobilization of AFP and CEA antibodies on epoxy functionalized monolith arrays. With a non-competitive immune response format, the presented AFP and CEA immune-affinity arrays were demonstrated as an inexpensive, flexible, homogeneous and stable array for detection of AFP and CEA.

Keywords: chemiluminescent detection, immune-affinity, monolithic copolymer array, UV-initiated copolymerization

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Authors: E. Marei, A. Hammad, S. Ismail, A. El-Gindy

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This study aims to investigate the ability of different formula of mixed bacteria as a biological treatments of wastewater after primary treatment as a bio-treatment and bio-removal and bio-adsorbent of different heavy metals in natural circumstances. The wastewater was collected from Sarpium forest site-Ismailia Governorate, Egypt. These treatments were mixture of free cells and mixture of immobilized cells of different bacteria. These different formulas of mixed bacteria were prepared under Lab. condition. The obtained data indicated that, as a result of wastewater bio-treatment, the removal rate was found to be 76.92 and 76.70% for biological oxygen demand, 79.78 and 71.07% for chemical oxygen demand, 32.45 and 36.84 % for ammonia nitrogen as well as 91.67 and 50.0% for phosphate after 24 and 28 hrs with mixed free cells and mixed immobilized cells, respectively. Moreover, the bio-removals of different heavy metals were found to reach 90.0 and 50. 0% for Cu ion, 98.0 and 98.5% for Fe ion, 97.0 and 99.3% for Mn ion, 90.0 and 90.0% Pb, 80.0% and 75.0% for Zn ion after 24 and 28 hrs with mixed free cells and mixed immobilized cells, respectively. The results indicated that 13.86 and 17.43% of removal efficiency and reduction of total dissolved solids were achieved after 24 and 28 hrs with mixed free cells and mixed immobilized cells, respectively.

Keywords: wastewater bio-treatment , bio-sorption heavy metals, biological desalination, immobilized bacteria, free cell bacteria

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Authors: Nisha Dhariwal, Anupama Sharma

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The synthesis of cellulose nanofibrils quaternized with 3‐chloro‐2‐hydroxypropyltrimethylammonium chloride (CHPTAC) in NaOH/urea aqueous solution has been reported. Xylenol Orange (XO) has been used as an indicator for selective detection of Sn (II) ions, by its immobilization on quaternized cellulose membrane. The effects of pH, reagent concentration and reaction time on the immobilization of XO have also been studied. The linear response, limit of detection, and interference of other metal ions have also been studied and no significant interference has been observed. The optical chemical sensor displayed good durability and short response time with negligible leaching of the reagent.

Keywords: cellulose, chemical sensor, heavy metal ions, indicator immobilization

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Authors: Norihiro Kato, Yuriko Takayama

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Some gram-negative bacteria enable the simultaneous activation of gene expression involved in N-acylhomoserine lactone (AHL) dependent cell-to-cell communication system. Such regulatory system for the bacterial group behavior is termed as quorum sensing (QS) because a diffusible AHL signal can accumulate around the cell during the increase of the cell density and trigger activation of the sequential QS process. By blocking the QS, the expression of diverse genes related to infection, antibiotic production, and biofilm formation is inhibited. Conditioning of QS by regulation of the DNA-receptor-AHL interaction is a potential target for enhancing host defenses against pathogenicity. We focused on engineered application of transcriptional regulator SpnR produced in opportunistic human pathogen Serratia marcescens. The SpnR can interact with AHL signals at an N-terminal domain and also with a promoter region of a QS target gene at a C-terminal domain. As the initial process of the QS activation, the SpnR forms a complex with the AHL to enhance the expression of pig cluster; the SpnR normally acts as an activator for the expression of the QS-dependent gene. In this research, we attempt to artificially control QS by changing the role of SpnR. The QS-dependent prodigiosin production is expected to inhibit by externally added SpnR in the culture broth of AS-1 strain because the AHL concentration was kept below the threshold by AHL-SpnR complex formation. Maltose-binding protein (MBP)-tagged SpnR (MBP-SpnR) was overexpressed in Escherichia coli and purified using an affinity chromatography equipped with an amylose resin column. The specific interaction between AHL and MBP-SpnR was demonstrated by quartz crystal microbalance (QCM) sensor. AHL with amino end-group was coupled with COOH-terminated self-assembled monolayer prepared on a gold electrode of 27-MHz quartz crystal sensor using water-soluble carbodiimide. After the injection of MBP-SpnR into a cup-type sensor cell filled with the buffer solution, time course of resonant frequency change (ΔFs) was determined. A decrease of ΔFs clearly showed the uptake of MBP-SpnR onto the AHL-immobilized electrode. Furthermore, no binding affinity was observed after the heat-inactivation of MBP-SpnR at 80ºC. These results suggest that MBP-SpnR possesses a specific affinity for AHL. MBP-SpnR was added to the culture medium as an AHL trap to study inhibitory effects on intracellularly accumulated prodigiosin. With approximately 2 µM MBP-SpnR, the amount of prodigiosin induced was half that of the control without any additives. In conclusion, the function of SpnR could be switched by adding it to the cell culture. Exogenously added MBP-SpnR possesses high affinity for AHL derived from cells and acts as an inhibitor of AHL-mediated QS.

Keywords: intracellular signaling, microbial biotechnology, quorum sensing, transcriptional regulator

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Authors: A. W. Zularisam, Lakhveer Singh, Mimi Sakinah Abdul Munaim

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In this study, the optimization of hydrogen production using polyethylene glycol (PEG) immobilized sludge was investigated in batch tests. Palm oil mill effluent (POME) is used as a substrate that can act as a carbon source. Experiment focus on the effect of some important affecting factors on fermentative hydrogen production. Results showed that immobilized sludge demonstrated the maximum hydrogen production rate of 340 mL/L-POME/h under follow optimal condition: amount of biomass 10 mg VSS/ g bead, PEG concentration 10%, and cell age 24 h or 40 h. More importantly, immobilized sludge not only enhanced hydrogen production but can also tolerate the harsh environment and produce hydrogen at the wide ranges of pH. The present results indicate the potential of PEG-immobilized sludge for large-scale operations as well; these factors play an important role in stable and continuous hydrogen production.

Keywords: bioydrogen, immobilization, polyethylene glycol, palm oil mill effluent, dark fermentation

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Authors: Sanja O. Podunavac-Kuzmanović, Lidija R. Jevrić, Strahinja Z. Kovačević

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In last decade, metal-organic complexes have captured intensive attention because of their wide range of biological activities such as antibacterial, antifungal, anticancerous, antimicrobial and antiHIV. Therefore, it is of great importance for the development of coordination chemistry to explore the assembly of functional organic ligands with metal ion and to investigate the relationship between the structure and property. In view of our studies, we reasoned that benzimidazoles complexed to metal ions could act as a potent antibacterial agents. Thus, we have bioassayed the inhibitory potency of benzimidazoles and their metal salts (Co or Ni) against Gram negative bacteria Escherichia coli. In order to validate our in vitro study, we performed in silico studies using molecular docking software’s. The investigated compounds and their metal complexes (Co, Ni) showed good antibacterial activity against Escherichia coli. In silico docking studies of the synthesized compounds suggested that complexed benzimidazoles have a greater binding affinity and enhanced antibacterial activity in comparison with noncomplexed ligands. In view of their enhanced inhibitory properties we propose that the studied complexes can be used as potential pharmaceuticals. This study is financially supported by COST action CM1306 and the project No. 114-451-347/2015-02, financially supported by the Provincial Secretariat for Science and Technological Development of Vojvodina.

Keywords: benzimidazoles, complexes, antibacterial, Escherichia coli, metal

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Authors: Saurabh B. Ganorkar, Atul A. Shirkhedkar

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The need for investigations protecting against toxic impurities though seems to be a primary requirement; the impurities which may prove non - toxic can be explored for their therapeutic potential if any to assist advanced drug discovery. The essential role of pharmaceutical analysis can thus be extended effectively to achieve it. The present study successfully achieved these objectives with characterization of major degradation products as impurities for Zileuton which has been used for to treat asthma since years. The forced degradation studies were performed to identify the potential degradation products using Ultra-fine Liquid-chromatography. Liquid-chromatography-Mass spectrometry (Time of Flight) and Proton Nuclear Magnetic Resonance Studies were utilized effectively to characterize the drug along with five major oxidative and hydrolytic degradation products (DP’s). The mass fragments were identified for Zileuton and path for the degradation was investigated. The characterized DP’s were subjected to In-Silico studies as XP Molecular Docking to compare the gain or loss in binding affinity with 5-Lipooxygenase enzyme. One of the impurity of was found to have the binding affinity more than the drug itself indicating for its potential to be more bioactive as better Antiasthmatic. The close structural resemblance has the ability to potentiate or reduce bioactivity and or toxicity. The chances of being active biologically at other sites cannot be denied and the same is achieved to some extent by predictions for probability of being active with Prediction of Activity Spectrum for Substances (PASS) The impurities found to be bio-active as Antineoplastic, Antiallergic, and inhibitors of Complement Factor D. The toxicological abilities as Ames-Mutagenicity, Carcinogenicity, Developmental Toxicity and Skin Irritancy were evaluated using Toxicity Prediction by Komputer Assisted Technology (TOPKAT). Two of the impurities were found to be non-toxic as compared to original drug Zileuton. As the drugs are purposed and repurposed effectively the impurities can also be; as they can have more binding affinity; less toxicity and better ability to be bio-active at other biological targets.

Keywords: UFLC, LC-MS-TOF, NMR, Zileuton, impurities, toxicity, bio-activity

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Authors: Marina Nisnevitch, Anton Valkov, Faina Nakonechny, Kate Adar Raik, Yamit Mualem

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Photosensitizers are dye compounds belonging to various chemical groups that in all the cases have a developed structure of conjugated double bonds. Under illumination with visible light, the photosensitizers are excited and transfer the absorbed energy to the oxygen dissolved in an aqueous phase, leading to production of a reactive oxygen species which cause irreversible damage to bacterial cells. When immobilized onto a solid phase, photosensitizers preserve their antibacterial properties. In the present study, photosensitizers were immobilized in polyethylene or propylene and tested for antimicrobial activity against Gram-positive S. aureus, S. epidermidis and Streptococcus sp. For this purpose, water-soluble photosensitizers, Rose Bengal sodium salt, and methylene blue as well as water-insoluble hematoporphyrin and Rose Bengal lactone, were immobilized by dissolution in melted polymers to yield 3 mm diameter rods and 3-5 mm beads. All four photosensitizers were found to be effective in the eradication of Gram-positive bacteria under illumination by a white luminescent lamp or sunlight. The immobilized photosensitizers can be applied for continuous water disinfection; they can be easily removed at the end of the treatment and reused.

Keywords: antimicrobial polymers, gram-positive bacteria, immobilization of photosensitizers, photodynamic antibacterial activity

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Authors: Manjusha C. Padole, Parag A. Deshpande

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Density functional theory calculations were carried out for identification of an active heterogeneous catalyst to carry out Heck coupling reaction which is of pharmaceutical importance. One of the carbonaceous nanomaterials, heterofullerene, was designed for the reaction. Stability and reactivity of the proposed heterofullerenes (C59M, M = Pd/Ni) were established with insights into the metal-carbon bond, electron affinity and chemical potential. Adsorbent potentials of both the heterofullerenes were examined from the adsorption study of four halobenzenes (C6H5F, C6H5Cl, C6H5Br and C6H5I). Oxidative addition activities of all four halobenzenes were investigated by developing free energy landscapes over both the heterofullerenes for rate determining step (oxidative addition). C6H5I showed a good catalytic activity for the rate determining step. Thus, C6H5I was proposed as a suitable halobenzene and complete free energy landscapes for Heck coupling reaction were developed over C59Pd and C59Ni. Smaller activation barriers observed over C59Pd in comparison with C59Ni put us in a position to propose C59Pd to be an efficient heterofullerene for carrying Heck coupling reaction.

Keywords: metal-substituted fullerene, density functional theory, electron affinity, oxidative addition, Heck coupling reaction

Procedia PDF Downloads 188

Authors: Furqan M. Kadhum, Asmaa A. Hussein, Maysaa Ch. Hatem

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Hyaluronidase enzyme was purified from the clinical isolate Staphyloccus aureus in three purification steps, first by precipitation with 90% saturated ammonium sulfate, ion exchange chromatography on DEAE-Cellulose, and gel filtration chromatography throughout Sephacryl S-300. Specific activity of the purified enzyme was reached 930 U/mg protein with 7.4 folds of purification and 46.5% recovery. The enzyme has an average molecular weight of about 69 kDa, with an optimum pH of enzyme activity and stability at pH 7, also the optimum temperature for activity was 37oC. The enzyme was stable with full activity at a temperature ranged between 30-40 oC. Metal ions showed variable inhibitory degree with the strongest effect for Fe+3, however, the chelating and reducing agents had no or little effects. Cytotoxic studies for purified and crude hyaluronidase against cancer cell Hep G2 type at different enzyme concentrations and exposure times showed that the inhibition effect of both crude and purified enzyme increased by increasing the enzyme concentration with no change was observed at 24hr, while at 48 and 72 hrs the same inhibition rate were observed for purified enzyme and differ for the crude filtrate.

Keywords: hyaluronidase, S. aureus, metal ions, cytotoxicity

Procedia PDF Downloads 415

Authors: Zeineb Seboui, Samar Dabbabi

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In this paper, we study the performance of Cu₂ZnSnS₄ (CZTS) based solar cell. In our knowledge, it is for the first time that the FTO/ZnO:Co/CZTS structure is simulated using the SILVACO-Atlas 2D simulation. Cu₂ZnSnS₄ (CZTS), ZnO:Co and FTO (SnO₂:F) layers have been deposited on glass substrates by the spray pyrolysis technique. The extracted physical properties, such as thickness and optical parameters of CZTS layer, are considered to create a new input data of CZTS based solar cell. The optimization of CZTS electron affinity and thickness is performed to have the best FTO/ZnO: Co/CZTS efficiency. The use of CZTS absorber layer with 3.99 eV electron affinity and 3.2 µm in thickness leads to the higher efficiency of 16.86 %, which is very important in the development of new technologies and new solar cell devices.

Keywords: CZTS solar cell, characterization, electron affinity, thickness, SILVACO-atlas 2D simulation

Procedia PDF Downloads 47

Authors: Misha Ali, Qayyum Husain, Nida Alam, Masood Ahmad

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Improving the activity and stability of the enzyme is an important aspect in bioremediation processes. Immobilization of enzyme is an efficient approach to amend the properties of biocatalyst required during wastewater treatment. The present study was done to immobilize partially purified ginger peroxidase on amino functionalized silica coated titanium dioxide nanocomposite. Interestingly there was an enhancement in enzyme activity after immobilization on nanosupport which was evident from effectiveness factor (η) value of 1.76. Immobilized enzyme was characterized by transmission electron microscopy, scanning electron microscopy and Fourier transform infrared spectroscopy. Immobilized peroxidase exhibited higher activity in a broad range of pH and temperature as compared to free enzyme. Also, the thermostability of peroxidase was strikingly improved upon immobilization. After six repeated uses, the immobilized peroxidase retained around 62% of its dye decolorization activity. There was a 4 fold increase in Vmax of immobilized peroxidase as compared to free enzyme. Circular dichroism spectroscopy demonstrated conformational changes in the secondary structure of enzyme, a possible reason for the enhanced enzyme activity after immobilization. Immobilized peroxidase was highly efficient in the removal of acid yellow 42 dye in a stirred batch process. Our study shows that this bio-remediating system has remarkable potential for treatment of aromatic pollutants present in wastewater.

Keywords: acid yellow 42, decolorization, ginger peroxidase, immobilization

Procedia PDF Downloads 215

Authors: Wei-Ting Kuo, Feng-Huei Lin

Abstract:

Epidermal growth factor receptor (EGFR) is often constitutively stimulated in cancer owing to the binding of ligands such as epidermal growth factor (EGF), so it is necessary to investigate the interaction between EGFR and its targeting biomolecules which were over ligands binding. This study would focus on the binding affinity and adhesion force of two targeting products anti-EGFR monoclonal antibody (mAb) and peptide A to EGFR comparing with EGF. Surface plasmon resonance (SPR) was used to obtain the equilibrium dissociation constant to evaluate the binding affinity. Atomic force microscopy (AFM) was performed to detect adhesion force. The result showed that binding affinity of mAb to EGFR was higher than that of EGF to EGFR, and peptide A to EGFR was lowest. The adhesion force between EGFR and mAb that was higher than EGF and peptide A to EGFR was lowest. From the studies, we could conclude that mAb had better adhesion force and binding affinity to EGFR than that of EGF and peptide A. SPR and AFM could confirm the interaction between receptor and targeting ligand easily and carefully. It provide a platform to screen ligands for receptor targeting and drug delivery.

Keywords: adhesion force, binding affinity, epidermal growth factor receptor, target molecule

Procedia PDF Downloads 403

Authors: A. Shahsavar, Z. Jakub

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Due to the outstanding properties of the 2D metal-organic frameworks (MOF), intensive computational and experimental studies have been done. However, the lack of fundamental studies of MOFs on the graphene backbone is observed. This work studies Fe and Ni as metal and tetracyanoquinodimethane (TCNQ) with a high electron affinity as an organic linker functionalized on graphene. Here we present DFT calculations results to unveil the electronic and magnetic properties of iron and nickel-TCNQ physisorbed on graphene. Adsorption and Fermi energies, structural, and magnetic properties will be reported. Our experimental observations prove Fe- and NiTCNQ@Gr/Ir(111) are thermally highly stable up to 500 and 250°C, respectively, making them promising materials for single-atom catalysts or high-density storage media.

Keywords: DFT, graphene, MTCNQ, self-assembly

Procedia PDF Downloads 75

Authors: Shaohua Li, Aihua Zhang, Kelly Zatopek, Saba Parvez, Andrew F. Gardner, Ivan R. Corrêa Jr., Christopher J. Noren, Ming-Qun Xu

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Covalent immobilization of enzymes on solid supports is an alternative approach to biocatalysis with the added benefits of simple enzyme removal, improved stability, and adaptability to automation and high-throughput applications. Nevertheless, immobilized enzymes generally suffer from reduced activities compared to their soluble counterparts. One major factor leading to activity loss is the intrinsic hydrophobic property of the supporting material surface, which could result in the conformational change/confinement of enzymes. We report a strategy of utilizing flexible poly (ethylene oxide) (PEO) moieties as to improve the surface hydrophilicity of solid supports used for enzyme immobilization. DNA modifying enzymes were covalently conjugated to PEO-coated magnetic-beads. Kinetics studies proved that the activities of the covalently-immobilized DNA modifying enzymes were greatly enhanced by the PEO modification on the bead surface.

Keywords: immobilized enzymes, biocatalysis, poly(ethylene oxide), surface modification

Procedia PDF Downloads 276

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

Procedia PDF Downloads 571

Authors: Khaled Alshammari

Abstract:

A facile approach to synthesizing highly active and stable Au/NiO catalysts for the hydrogenation of nitro-aromatics is reported. Preformed gold nanoparticles have been immobilized onto NiO using a colloidal method. In this article, the reduction of 4-nitrophenol with NaBH4 has been used as a model reaction to investigate the catalytic activity of synthesized Au/NiO catalysts. In addition, we report a systematic study of the reduction kinetics and the influence of specific reaction parameters such as (i) temperature, (ii) stirring rate, (iii) sodium borohydride concentration and (iv) substrate/metal molar ratio. The reaction has been performed at a substrate/metal molar ratio of 7.4, a ratio significantly higher than previously reported. The reusability of the catalyst has been examined, with little to no decrease in activity observed over 5 catalytic cycles. Systematic variation of Au loading reveals the successful synthesis of low-cost and efficient Au/NiO catalysts at very low Au content and using high substrate/metal molar ratios.

Keywords: nonochemistry, catalyst, nanoparticles supported, characterization of materials, colloidal nanoparticles

Procedia PDF Downloads 18