Cellulose acetate membranes functionalized with resveratrol by covalent immobilization for improved osseointegration

Science.gov (United States)

Pandele, A. M.; Neacsu, P.; Cimpean, A.; Staras, A. I.; Miculescu, F.; Iordache, A.; Voicu, S. I.; Thakur, V. K.; Toader, O. D.

2018-04-01

Covalent immobilization of resveratrol onto cellulose acetate polymeric membranes used as coating on a Mg-1Ca-0.2Mn-0.6Zr alloy is presented for potential application in the improvement of osseointegration processes. For this purpose, cellulose acetate membrane is hydrolysed in the presence of potassium hydroxide, followed by covalent immobilization of aminopropyl triethoxy silane. Resveratrol was immobilized onto membranes using glutaraldehyde as linker. The newly synthesised functional membranes were thoroughly characterized for their structural characteristics determination employing X-ray photoelectron spectroscopy (XPS), infrared spectroscopy (FT-IR), Raman spectroscopy, thermogravimetric analysis (TGA/DTG) and scanning electron microscopy (SEM) techniques. Subsequently, in vitro cellular tests were performed for evaluating the cytotoxicity biocompatibility of synthesized materials and also the osseointegration potential of obtained derivatised membrane material. It was demonstrated that both polymeric membranes support viability and proliferation of the pre-osteoblastic MC3T3-E1 cells, thus providing a good protection against the potential harmful effects of the compounds released from coated alloys. Furthermore, cellulose acetate membrane functionalized with resveratrol exhibits a significant increase in alkaline phosphatase activity and extracellular matrix mineralization, suggesting its suitability to function as an implant surface coating for guided bone regeneration.

Carbon paste electrode with covalently immobilized thionine for electrochemical sensing of hydrogen peroxide

Science.gov (United States)

Thenmozhi, K.; Sriman Narayanan, S.

2017-11-01

A water-soluble redox mediator, thionin was covalently immobilized to the functionalized graphite powder and a carbon paste electrode was fabricated from this modified graphite powder. The immobilization procedure proved to be effective in anchoring the thionin mediator in the graphite electrode setup without any leakage problem during the electrochemical studies. The covalent immobilization of the thionin mediator was studied with FT-IR and the electrochemical response of the thionin carbon paste electrode was optimized on varying the supporting electrolyte, pH and scan rate. The modified electrode exhibited well-defined electrocatalytic activity towards the reduction of H2O2 at a lower potential of -0.266 V with good sensitivity. The developed amperometric sensor was efficient towards H2O2 in the linear range from 2.46 × 10-5 M to 4.76 × 10-3 M, with a detection limit of 1.47 × 10-5 M respectively. Important advantages of this sensor are its excellent electrochemical performance, simple fabrication, easy renewability, reproducible analytical results, acceptable accuracy and good operational and long-term stability.

Covalent immobilization of redox protein within the mesopores of transparent conducting electrodes

Czech Academy of Sciences Publication Activity Database

Müller, V.; Rathouský, Jiří; Fattakhova-Rohlfing, D.

2014-01-01

Roč. 116, JAN 2014 (2014), s. 1-8 ISSN 0013-4686 R&D Projects: GA ČR GA104/08/0435 Institutional support: RVO:61388955 Keywords : Covalent immobilization * Porous electrodes * Redox proteins Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 4.504, year: 2014

Efficient protein immobilization on polyethersolfone electrospun nanofibrous membrane via covalent binding for biosensing applications

Energy Technology Data Exchange (ETDEWEB)

Mahmoudifard, Matin [Institute for Nanoscience and Nanotechnology, Sharif University of Technology, Tehran (Iran, Islamic Republic of); Soudi, Sara [Stem Cell Biology Department, Stem Cell Technology Research Center, Tehran (Iran, Islamic Republic of); Department of Immunology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran (Iran, Islamic Republic of); Soleimani, Masoud [Department of Hematology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran (Iran, Islamic Republic of); Hosseinzadeh, Simzar [Nanotechnology and Tissue Engineering Department, Stem Cell Technology Research Center, Tehran (Iran, Islamic Republic of); School of Advanced Medical Technologies, Tehran University of Medical Sciences, Tehran (Iran, Islamic Republic of); Esmaeili, Elaheh [Institute for Nanoscience and Nanotechnology, Sharif University of Technology, Tehran (Iran, Islamic Republic of); Department of Hematology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran (Iran, Islamic Republic of); Vossoughi, Manouchehr, E-mail: vosoughi@sharif.edu [Institute for Nanoscience and Nanotechnology, Sharif University of Technology, Tehran (Iran, Islamic Republic of); Chemical and Petroleum Engineering Department, Sharif University of Technology, Tehran (Iran, Islamic Republic of)

2016-01-01

In this paper we introduce novel strategy for antibody immobilization using high surface area electrospun nanofibrous membrane based on ethyl-3-(3-dimethylaminopropyl)-carbodiimide/N-hydroxysuccinimide (EDC/NHS) coupling chemistry. To present the high performance of proposed biosensors, anti-staphylococcus enterotoxin B (anti-SEB) was used as a model to demonstrate the utility of our proposed system. Polymer solution of polyethersolfone was used to fabricate fine nanofibrous membrane. Moreover, industrial polyvinylidene fluoride membrane and conventional microtiter plate were also used to compare the efficiency of antibody immobilization. Scanning electron microscopy images were taken to study the morphology of the membranes. The surface activation of nanofibrous membrane was done with the help of O{sub 2} plasma. PES nanofibrous membrane with carboxyl functional groups for covalent attachment of antibodies were treated by EDC/NHS coupling agent. The quantity of antibody immobilization was measured by enzyme-linked immuno sorbent assay (ELISA) method. Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) spectroscopy was performed to confirm the covalent immobilization of antibody on membrane. Atomic force microscopy, scanning electron microscopy and invert fluorescence microscopy were used to analyze the antibody distribution pattern on solid surfaces. Results show that oxygen plasma treatment effectively increased the amount of antibody immobilization through EDC/NHS coupling chemistry. It was found that the use of nanofibrous membrane causes the improved detection signal of ELISA based biosensors in comparison to the standard assay carried out in the 96-well microtiter plate. This method has the potential to improve the ELISA-based biosensor and we believe that this technique can be used in various biosensing methods. - Highlights: • Introduction of novel strategy for antibody immobilization using high surface area electrospun

Covalent immobilization of penicillin G acylase on aminopropyl-functionalized mesostructured cellular foams.

Science.gov (United States)

Zhao, Junqi; Wang, Yujun; Luo, Guangsheng; Zhu, Shenlin

2010-10-01

Mesostructured cellular foams (MCFs) are suitable for biomolecular immobilization because of their relatively large-pore diameter and pore volume. Penicillin G acylase (PGA) was immobilized on aminopropyl-functionalized MCFs through Schiff base reaction. It is shown that PGA could be fixed more firmly through the covalent immobilization on aminopropyl-functionalized MCFs support than through the adsorption immobilization on blank MCFs. The PGA loading amount on the aminopropyl-functionalized MCFs could reach 443 mg/g (dry support), and the apparent activity could achieve up to 4138 U/g (dry support). The influence of the amount of grafted aminopropyl group was studied, and it is found that the optimal molar ratio of MCFs to APTS was 15/1; in addition, the suitable enzyme distribution density for the specific activity of the immobilized PGA was 0.7 mg enzyme per m(2) of specific area of MCFs. Copyright 2010 Elsevier Ltd. All rights reserved.

Covalent Immobilization of Bacillus licheniformis γ-Glutamyl Transpeptidase on Aldehyde-Functionalized Magnetic Nanoparticles

Directory of Open Access Journals (Sweden)

Meng-Chun Chi

2013-02-01

Full Text Available This work presents the synthesis and use of surface-modified iron oxide nanoparticles for the covalent immobilization of Bacillus licheniformis γ-glutamyl transpeptidase (BlGGT. Magnetic nanoparticles were prepared by an alkaline solution of divalent and trivalent iron ions, and they were subsequently treated with 3-aminopropyltriethoxysilane (APES to obtain the aminosilane-coated nanoparticles. The functional group on the particle surface and the amino group of BlGGT was then cross-linked using glutaraldehyde as the coupling reagent. The loading capacity of the prepared nanoparticles for BlGGT was 34.2 mg/g support, corresponding to 52.4% recovery of the initial activity. Monographs of transmission electron microscopy revealed that the synthesized nanoparticles had a mean diameter of 15.1 ± 3.7 nm, and the covalent cross-linking of the enzyme did not significantly change their particle size. Fourier transform infrared spectroscopy confirmed the immobilization of BlGGT on the magnetic nanoparticles. The chemical and kinetic behaviors of immobilized BlGGT are mostly consistent with those of the free enzyme. The immobilized enzyme could be recycled ten times with 36.2% retention of the initial activity and had a comparable stability respective to free enzyme during the storage period of 30 days. Collectively, the straightforward synthesis of aldehyde-functionalized nanoparticles and the efficiency of enzyme immobilization offer wide perspectives for the practical use of surface-bound BlGGT.

Potential Applications of Carbohydrases Immobilization in the Food Industry

Science.gov (United States)

Contesini, Fabiano Jares; de Alencar Figueira, Joelise; Kawaguti, Haroldo Yukio; de Barros Fernandes, Pedro Carlos; de Oliveira Carvalho, Patrícia; Nascimento, Maria da Graça; Sato, Hélia Harumi

2013-01-01

Carbohydrases find a wide application in industrial processes and products, mainly in the food industry. With these enzymes, it is possible to obtain different types of sugar syrups (viz. glucose, fructose and inverted sugar syrups), prebiotics (viz. galactooligossacharides and fructooligossacharides) and isomaltulose, which is an interesting sweetener substitute for sucrose to improve the sensory properties of juices and wines and to reduce lactose in milk. The most important carbohydrases to accomplish these goals are of microbial origin and include amylases (α-amylases and glucoamylases), invertases, inulinases, galactosidases, glucosidases, fructosyltransferases, pectinases and glucosyltransferases. Yet, for all these processes to be cost-effective for industrial application, a very efficient, simple and cheap immobilization technique is required. Immobilization techniques can involve adsorption, entrapment or covalent bonding of the enzyme into an insoluble support, or carrier-free methods, usually based on the formation of cross-linked enzyme aggregates (CLEAs). They include a broad variety of supports, such as magnetic materials, gums, gels, synthetic polymers and ionic resins. All these techniques present advantages and disadvantages and several parameters must be considered. In this work, the most recent and important studies on the immobilization of carbohydrases with potential application in the food industry are reviewed. PMID:23344046

Enhanced Biological Response of AVS-Functionalized Ti-6Al-4V Alloy through Covalent Immobilization of Collagen.

Science.gov (United States)

Rezvanian, Parsa; Daza, Rafael; López, Patricia A; Ramos, Milagros; González-Nieto, Daniel; Elices, Manuel; Guinea, Gustavo V; Pérez-Rigueiro, José

2018-02-20

This study presents the development of an efficient procedure for covalently immobilizing collagen molecules on AVS-functionalized Ti-6Al-4V samples, and the assessment of the survival and proliferation of cells cultured on these substrates. Activated Vapor Silanization (AVS) is a versatile functionalization technique that allows obtaining a high density of active amine groups on the surface. A procedure is presented to covalently bind collagen to the functional layer using EDC/NHS as cross-linker. The covalently bound collagen proteins are characterized by fluorescence microscopy and atomic force microscopy and their stability is tested. The effect of the cross-linker concentration on the process is assessed. The concentration of the cross-linker is optimized and a reliable cleaning protocol is developed for the removal of the excess of carbodiimide from the samples. The results demonstrate that the covalent immobilization of collagen type I on Ti-6Al-4V substrates, using the optimized protocol, increases the number of viable cells present on the material. Consequently, AVS in combination with the carbodiimide chemistry appears as a robust method for the immobilization of proteins and, for the first time, it is shown that it can be used to enhance the biological response to the material.

Covalent Immobilization of β-Glucosidase on Magnetic Particles for Lignocellulose Hydrolysis

DEFF Research Database (Denmark)

Alftrén, Johan; Hobley, Timothy John

2013-01-01

β-Glucosidase hydrolyzes cellobiose to glucose and is an important enzyme in the consortium used for hydrolysis of cellulosic and lignocellulosic feedstocks. In the present work, β-glucosidase was covalently immobilized on non-porous magnetic particles to enable re-use of the enzyme. It was found...... that particles activated with cyanuric chloride and polyglutaraldehyde gave the highest bead-related immobilized enzyme activity when tested with p-nitrophenyl-β-D-glucopyranoside (104.7 and 82.2 U/g particles, respectively). Furthermore, the purified β-glucosidase preparation from Megazyme gave higher bead......-related enzyme activities compared to Novozym 188 (79.0 and 9.8 U/g particles, respectively). A significant improvement in thermal stability was observed for immobilized enzyme compared to free enzyme; after 5 h (at 65 °C), 36 % of activity remained for the former, while there was no activity in the latter...

Covalent immobilization of β-glucosidase on magnetic particles for lignocellulose hydrolysis.

Science.gov (United States)

Alftrén, Johan; Hobley, Timothy John

2013-04-01

β-Glucosidase hydrolyzes cellobiose to glucose and is an important enzyme in the consortium used for hydrolysis of cellulosic and lignocellulosic feedstocks. In the present work, β-glucosidase was covalently immobilized on non-porous magnetic particles to enable re-use of the enzyme. It was found that particles activated with cyanuric chloride and polyglutaraldehyde gave the highest bead-related immobilized enzyme activity when tested with p-nitrophenyl-β-D-glucopyranoside (104.7 and 82.2 U/g particles, respectively). Furthermore, the purified β-glucosidase preparation from Megazyme gave higher bead-related enzyme activities compared to Novozym 188 (79.0 and 9.8 U/g particles, respectively). A significant improvement in thermal stability was observed for immobilized enzyme compared to free enzyme; after 5 h (at 65 °C), 36 % of activity remained for the former, while there was no activity in the latter. The performance and recyclability of immobilized β-glucosidase on more complex substrate (pretreated spruce) was also studied. It was shown that adding immobilized β-glucosidase (16 U/g dry matter) to free cellulases (8 FPU/g dry matter) increased the hydrolysis yield of pretreated spruce from ca. 44 % to ca. 65 %. In addition, it was possible to re-use the immobilized β-glucosidase in the spruce and retain activity for at least four cycles. The immobilized enzyme thus shows promise for lignocellulose hydrolysis.

Improving the Thermostability and Optimal Temperature of a Lipase from the Hyperthermophilic Archaeon Pyrococcus furiosus by Covalent Immobilization

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Roberta V. Branco

2015-01-01

Full Text Available A recombinant thermostable lipase (Pf2001Δ60 from the hyperthermophilic Archaeon Pyrococcus furiosus (PFUL was immobilized by hydrophobic interaction on octyl-agarose (octyl PFUL and by covalent bond on aldehyde activated-agarose in the presence of DTT at pH = 7.0 (one-point covalent attachment (glyoxyl-DTT PFUL and on glyoxyl-agarose at pH 10.2 (multipoint covalent attachment (glyoxyl PFUL. The enzyme’s properties, such as optimal temperature and pH, thermostability, and selectivity, were improved by covalent immobilization. The highest enzyme stability at 70°C for 48 h incubation was achieved for glyoxyl PFUL (around 82% of residual activity, whereas glyoxyl-DTT PFUL maintained around 69% activity, followed by octyl PFUL (27% remaining activity. Immobilization on glyoxyl-agarose improved the optimal temperature to 90°C, while the optimal temperature of octyl PFUL was 70°C. Also, very significant changes in activity with different substrates were found. In general, the covalent bond derivatives were more active than octyl PFUL. The E value also depended substantially on the derivative and the conditions used. It was observed that the reaction of glyoxyl-DTT PFUL using methyl mandelate as a substrate at pH 7 presented the best results for enantioselectivity E=22 and enantiomeric excess (ee (% = 91.

Co-expression of Exo-inulinase and Endo-inulinase Genes in the Oleaginous Yeast Yarrowia lipolytica for Efficient Single Cell Oil Production from Inulin.

Science.gov (United States)

Shi, Nianci; Mao, Weian; He, Xiaoxia; Chi, Zhe; Chi, Zhenming; Liu, Guanglei

2018-05-01

Yarrowia lipolytica is a promising platform for the single cell oil (SCO) production. In this study, a transformant X+N8 in which exo- and endo-inulinase genes were co-expressed could produce an inulinase activity of 124.33 U/mL within 72 h. However, the inulinase activity of a transformant X2 carrying a single exo-inulinase gene was only 47.33 U/mL within 72 h. Moreover, the transformant X+N8 could accumulate 48.13% (w/w) SCO from inulin and the cell dry weight reached 13.63 g/L within 78 h, which were significantly higher than those of the transformant X2 (41.87% (w/w) and 11.23 g/L) under the same conditions. In addition, inulin hydrolysis and utilization of the transformant X+N8 were also more efficient than those of the transformant X2 during the fermentation process. These results demonstrated that the co-expression of the exo- and endo-inulinase genes significantly enhanced the SCO production from inulin due to the improvement of the inulinase activity and the synergistic action of exo- and endo-inulinase. Besides, over 95.01% of the fatty acids from the transformant X+N8 were C16-C18, especially C18:1 (53.10%), suggesting that the fatty acids could be used as feedstock for biodiesel production.

NOVEL APPLICATION OF POROUS AND CELLULAR MATERIALS FOR COVALENT IMMOBILIZATION OF PEPSIN

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K. Szałapata

Full Text Available Abstract Pepsin was immobilized via covalent bonds on different carriers: a silica gel carrier, acrylic beads, and a cellulose-based carrier - Granocel. All carriers were functionalized through the presence of -OH, -COOH, -NH2, or glycidyl groups on their surfaces. Three different cross-linkers were used for activation thereof. The results showed that Granocel activated by glutaraldehyde or carbodiimide and silica gel activated by glutaraldehyde were suitable carriers for the expression of enzyme activity. The optimum pH range for the native enzyme was 2.5-3.5 and this range was extended to the value 6.5 in the case of enzyme immobilized on the silica gel carrier and on Granocel. The optimum temperature values for the native and immobilized enzyme were in the range 37-40 °C and 40-50 °C, respectively. The activity of the immobilized pepsin at different values of pH and temperature was higher in comparison with the activity of the free enzyme.

Covalent Immobilization of Cellulase Using Magnetic Poly(ionic liquid) Support: Improvement of the Enzyme Activity and Stability.

Science.gov (United States)

Hosseini, Seyed Hassan; Hosseini, Seyedeh Ameneh; Zohreh, Nasrin; Yaghoubi, Mahshid; Pourjavadi, Ali

2018-01-31

A magnetic nanocomposite was prepared by entrapment of Fe 3 O 4 nanoparticles into the cross-linked ionic liquid/epoxy type polymer. The resulting support was used for covalent immobilization of cellulase through the reaction with epoxy groups. The ionic surface of the support improved the adsorption of enzyme, and a large amount of enzyme (106.1 mg/g) was loaded onto the support surface. The effect of the presence of ionic monomer and covalent binding of enzyme was also investigated. The structure of support was characterized by various instruments such as FT-IR, TGA, VSM, XRD, TEM, SEM, and DLS. The activity and stability of immobilized cellulase were investigated in the prepared support. The results showed that the ionic surface and covalent binding of enzyme onto the support improved the activity, thermal stability, and reusability of cellulase compared to free cellulase.

Inulinase production in a packed bed reactor by solid state fermentation.

Science.gov (United States)

Dilipkumar, M; Rajamohan, N; Rajasimman, M

2013-07-01

In this work, production of inulinase was carried out in a packed bed reactor (PBR) under solid state fermentation. Kluyveromyces marxianus var. marxianus was used to produce the inulinase using pressmud as substrate. The parameters like air flow rate, packing density and particle size were optimized using response surface methodology (RSM) to maximize the inulinase production. The optimum conditions for the maximum inulinase production were: air flow rate - 0.82 L/min, packing density - 40 g/L and particle size - 0.0044 mm (mesh - 14/20). At these optimized conditions, the production of inulinase was found to be 300.5 unit/gram of dry substrate (U/gds). Copyright © 2013 Elsevier Ltd. All rights reserved.

Improved immobilization of laccase on a glassy carbon electrode by oriented covalent attachment

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Liu Xin

2014-01-01

Full Text Available A laccase from Thermus thermophilus HB27 was reported to be potentially useful in the design of a temperature controlled biofuel cell. For enhancing its application in different thermal conditions, we engineered a laccase-oriented immobilized electrode. A site-directed mutant N323C of the laccase was constructed. A photometric assay was employed in order to compare the catalytic properties of wild-type laccase and mutant. The mutant was attached to a glass carbon electrode by covalent cross-linking. The electrochemical properties of the immobilized laccase were investigated by cyclic voltammetry. This immobilization allowed the active electrode to function at temperatures up to 95°C. The thermal and pH dependence profiles were similar to those of the soluble enzyme investigated by spectrophotometry.

Protein covalent immobilization via its scarce thiol versus abundant amine groups: Effect on orientation, cell binding domain exposure and conformational lability.

Science.gov (United States)

Ba, O M; Hindie, M; Marmey, P; Gallet, O; Anselme, K; Ponche, A; Duncan, A C

2015-10-01

Quantity, orientation, conformation and covalent linkage of naturally cell adhesive proteins adsorbed or covalently linked to a surface, are known to influence the preservation of their subsequent long term cell adhesion properties and bioactivity. In the present work, we explore two different strategies for the covalent linking of plasma fibronectin (pFN) - used as a cell adhesive model protein, onto a polystyrene (PS) surface. One is aimed at tethering the protein to the surface in a semi-oriented fashion (via one of the 4 free thiol reactive groups on the protein) with a heterofunctional coupling agent (SSMPB method). The other aims to immobilize the protein in a more random fashion by reaction between the abundant pendant primary amine bearing amino acids of the pFN and activated carboxylic surface functions obtained after glutaric anhydride surface treatment (GA method). The overall goal will be to verify the hypothesis of a correlation between covalent immobilization of a model cell adhesive protein to a PS surface in a semi-oriented configuration (versus randomly oriented) with promotion of enhanced exposure of the protein's cell binding domain. This in turn would lead to enhanced cell adhesion. Ideally the goal is to elaborate substrates exhibiting a long term stable protein monolayer with preserved cell adhesive properties and bioactivity for biomaterial and/or cell adhesion commercial plate applications. However, the initial restrictive objective of this paper is to first quantitatively and qualitatively investigate the reversibly (merely adsorbed) versus covalently irreversibly bound protein to the surface after the immobilization procedure. Although immobilized surface amounts were similar (close to the monolayer range) for all immobilization approaches, covalent grafting showed improved retention and stronger "tethering" of the pFN protein to the surface (roughly 40%) after SDS rinsing compared to that for mere adsorption (0%) suggesting an added value

Production, characterization and application of inulinase from fungal endophyte CCMB 328

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Diego S. Nascimento

2012-06-01

Full Text Available Inulinase (β-2,1-D- fructan fructanohydrolase, EC 3.2.1.7, targets the β-2,1 linkage of inulin, a polyfructan consisting of linear β-2,1 linked fructose, and hydrolyzes it into fructose. This use provides an alternative to produce fructose syrup through the hydrolysis of inulin. The objective of this work was to study the production, characterization and applications of inulinases from the fungal endophyte CCMB 328 isolated from the Brazilian semi-arid region. Response Surface Methodology (RSM was employed to evaluate the effect of variables (concentration of glucose and yeast extract, on secreted inulinase activities detected in the culture medium and also in the inulin hydrolysis. The results showed that the best conditions for inulinase production by CCMB 328 are 9.89 g / L for glucose and 1.09 g / L for yeast extract. The concentration of 0.20 mol/L of NaCl and KCl increased the activity of inulinase from CCMB 328 by approximately 63% and 37%, respectively. The results also showed that the inulinase has potential for inulin hydrolysis, whose conversion yields roughly 72.48 % for an initial concentration of inulin at 1% (w/v.A enzima inulinase (EC 3.2.1.7, β-D-frutano frutanohidrolase atua sobre as ligações β-2,1 da inulina, um polifrutano consistindo de frutose unida por ligações β-2,1. A hidrólise de inulina através do uso de inulinase é uma alternativa viável para a obtenção de xarope de frutose. O objetivo deste trabalho foi estudar a produção, caracterização e aplicação de inulinase obtidas a partir do fungo endofítico CCMB 328, isolado do semi-árido brasileiro. A metodologia de Superfície de Resposta (MSR foi empregado para avaliar os efeitos das variáveis (concentração de glicose e extrato de levedura na atividade da enzima inulinase produzida em meio de cultura líquido e também para avaliar a atividade da enzima na hidrólise de inulina. Os resultados mostraram que as melhores condições para a produ

A simple approach for immobilization of gold nanoparticles on graphene oxide sheets by covalent bonding

NARCIS (Netherlands)

Pham, Tuan Anh; Choi, Byung Choon; Lim, Kwon Taek; Jeong, Yeon Tae

2011-01-01

Amino - functionalized gold nanoparticles with a diameter of around 5 nm were immobilized onto the surface of graphene oxide sheets (GOS) by covalent bonding through a simple amidation reaction. Pristine graphite was firstly oxidized and exfoliated to obtain GOS, which further were acylated with

Electrochemical Biosensor for Nitrite Based on Polyacrylic-Graphene Composite Film with Covalently Immobilized Hemoglobin

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Raja Zaidatul Akhmar Raja Jamaluddin

2018-04-01

Full Text Available A new biosensor for the analysis of nitrite in food was developed based on hemoglobin (Hb covalently immobilized on the succinimide functionalized poly(n-butyl acrylate-graphene [poly(nBA-rGO] composite film deposited on a carbon-paste screen-printed electrode (SPE. The immobilized Hb on the poly(nBA-rGO conducting matrix exhibited electrocatalytic ability for the reduction of nitrite with significant enhancement in the reduction peak at −0.6 V versus Ag/AgCl reference electrode. Thus, direct determination of nitrite can be achieved by monitoring the cathodic peak current signal of the proposed polyacrylic-graphene hybrid film-based voltammetric nitrite biosensor. The nitrite biosensor exhibited a reproducible dynamic linear response range from 0.05–5 mg L−1 nitrite and a detection limit of 0.03 mg L−1. No significant interference was observed by potential interfering ions such as Ca2+, Na+, K+, NH4+, Mg2+, and NO3− ions. Analysis of nitrite in both raw and processed edible bird’s nest (EBN samples demonstrated recovery of close to 100%. The covalent immobilization of Hb on poly(nBA-rGO composite film has improved the performance of the electrochemical nitrite biosensor in terms of broader detection range, lower detection limit, and prolonged biosensor stability.

Green coconut fiber: a novel carrier for the immobilization of commercial laccase by covalent attachment for textile dyes decolourization.

Science.gov (United States)

Cristóvão, Raquel O; Silvério, Sara C; Tavares, Ana P M; Brígida, Ana Iraidy S; Loureiro, José M; Boaventura, Rui A R; Macedo, Eugénia A; Coelho, Maria Alice Z

2012-09-01

Commercial laccase formulation was immobilized on modified green coconut fiber silanized with 3-glycidoxypropyltrimethoxysilane, aiming to achieve a cheap and effective biocatalyst. Two different strategies were followed: one point (pH 7.0) and multipoint (pH 10.0) covalent attachment. The influence of immobilization time on enzymatic activity and the final reduction with sodium borohydride were evaluated. The highest activities were achieved after 2 h of contact time in all situations. Commercial laccase immobilized at pH 7.0 was found to have higher activity and higher affinity to the substrate. However, the immobilization by multipoint covalent attachment improved the biocatalyst thermal stability at 50 °C, when compared to soluble enzyme and to the immobilized enzyme at pH 7.0. The Schiff's bases reduction by sodium borohydride, in spite of causing a decrease in enzyme activity, showed to contribute to the increase of operational stability through bonds stabilization. Finally, these immobilized enzymes showed high efficiency in the continuous decolourization of reactive textile dyes. In the first cycle, the decolourization is mainly due to dyes adsorption on the support. However, when working in successive cycles, the adsorption capacity of the support decreases (saturation) and the enzymatic action increases, indicating the applicability of this biocatalyst for textile wastewater treatment.

Recent advances in covalent, site-specific protein immobilization [version 1; referees: 3 approved

Directory of Open Access Journals (Sweden)

Morten Meldal

2016-09-01

Full Text Available The properties of biosensors, biomedical implants, and other materials based on immobilized proteins greatly depend on the method employed to couple the protein molecules to their solid support. Covalent, site-specific immobilization strategies are robust and can provide the level of control that is desired in this kind of application. Recent advances include the use of enzymes, such as sortase A, to couple proteins in a site-specific manner to materials such as microbeads, glass, and hydrogels. Also, self-labeling tags such as the SNAP-tag can be employed. Last but not least, chemical approaches based on bioorthogonal reactions, like the azide–alkyne cycloaddition, have proven to be powerful tools. The lack of comparative studies and quantitative analysis of these immobilization methods hampers the selection process of the optimal strategy for a given application. However, besides immobilization efficiency, the freedom in selecting the site of conjugation and the size of the conjugation tag and the researcher’s expertise regarding molecular biology and/or chemical techniques will be determining factors in this regard.

Epoxy-functionalized mesostructured cellular foams as effective support for covalent immobilization of penicillin G acylase

Science.gov (United States)

Xue, Ping; Xu, Fang; Xu, Lidong

2008-12-01

The epoxy-functionalized mesoporous cellular foams (G-MCFs) with high specific surface area (˜400 m 2/g) and large-size mesopores (˜17 nm) were obtained by condensation of 3-glycidoxypropyltriethoxysilane (GPTS) and the surface silanol groups of mesoporous cellular foams (MCFs) and used as the support for immobilization of penicillin G acylase (PGA). The structural properties of G-MCF were characterized by FT-IR, N 2 adsorption, TG-DTA and 29Si MAS NMR. The studies indicated that the glycidoxypropyl groups were chemically bonded to the silicon atoms on the surface of MCF. The epoxy-functionalized mesoporous cellular foams can provide the microenvironments suitable for the immobilization of PGA, and the enzyme molecules could be immobilized covalently onto the G-MCF under mild conditions by reaction between the amino groups of the enzyme molecules and the epoxy groups on the surface of G-MCF. The PGA immobilized on G-MCF (PGA/G-MCF) exhibited the apparent activity of 1782 IU/g and 46.6% of activity recovery for hydrolyzing penicillin G potassium to produce 6-aminopenicillanic acid at 37 °C which were higher than that of PGA on pure silica MCF (1521 IU/g and 39.8%, respectively). The kinetic study also indicated that PGA immobilized on G-MCF has a Km of 2.1 × 10 -2 mol/L lower than that of PGA immobilized on the pure silica MCF (5.0 × 10 -2 mol/L). These may be attributed to the enhanced surface affinity between G-MCF support and the substrate molecules. Due to the covalent immobilization of PGA molecules on the surface of G-MCF, the immobilized PGA with considerable operational stability was achieved. The activity of PGA/G-MCF is still about 91.4% of its initial activity at the 10th cycle reuse while that of PGA/MCF only remains 41.5% of its initial activity at the same reuse numbers. In addition, the investigation results show the thermal stability and durability on acid or basic medium of PGA immobilized on G-MCF were improved remarkably.

Epoxy-functionalized mesostructured cellular foams as effective support for covalent immobilization of penicillin G acylase

International Nuclear Information System (INIS)

Xue Ping; Xu Fang; Xu Lidong

2008-01-01

The epoxy-functionalized mesoporous cellular foams (G-MCFs) with high specific surface area (∼400 m 2 /g) and large-size mesopores (∼17 nm) were obtained by condensation of 3-glycidoxypropyltriethoxysilane (GPTS) and the surface silanol groups of mesoporous cellular foams (MCFs) and used as the support for immobilization of penicillin G acylase (PGA). The structural properties of G-MCF were characterized by FT-IR, N 2 adsorption, TG-DTA and 29 Si MAS NMR. The studies indicated that the glycidoxypropyl groups were chemically bonded to the silicon atoms on the surface of MCF. The epoxy-functionalized mesoporous cellular foams can provide the microenvironments suitable for the immobilization of PGA, and the enzyme molecules could be immobilized covalently onto the G-MCF under mild conditions by reaction between the amino groups of the enzyme molecules and the epoxy groups on the surface of G-MCF. The PGA immobilized on G-MCF (PGA/G-MCF) exhibited the apparent activity of 1782 IU/g and 46.6% of activity recovery for hydrolyzing penicillin G potassium to produce 6-aminopenicillanic acid at 37 o C which were higher than that of PGA on pure silica MCF (1521 IU/g and 39.8%, respectively). The kinetic study also indicated that PGA immobilized on G-MCF has a K m of 2.1 x 10 -2 mol/L lower than that of PGA immobilized on the pure silica MCF (5.0 x 10 -2 mol/L). These may be attributed to the enhanced surface affinity between G-MCF support and the substrate molecules. Due to the covalent immobilization of PGA molecules on the surface of G-MCF, the immobilized PGA with considerable operational stability was achieved. The activity of PGA/G-MCF is still about 91.4% of its initial activity at the 10th cycle reuse while that of PGA/MCF only remains 41.5% of its initial activity at the same reuse numbers. In addition, the investigation results show the thermal stability and durability on acid or basic medium of PGA immobilized on G-MCF were improved remarkably.

Epoxy-functionalized mesostructured cellular foams as effective support for covalent immobilization of penicillin G acylase

Energy Technology Data Exchange (ETDEWEB)

Xue Ping [Key Laboratory of Energy Resources and Chemical Engineering, Ningxia University, Yinchuan 750021 (China)], E-mail: Ping@nxu.edu.cn; Xu Fang [Department of Molecule Biology, Ningxia Medical College, Yinchuan 750021 (China); Xu Lidong [Key Laboratory of Energy Resources and Chemical Engineering, Ningxia University, Yinchuan 750021 (China)

2008-12-30

The epoxy-functionalized mesoporous cellular foams (G-MCFs) with high specific surface area ({approx}400 m{sup 2}/g) and large-size mesopores ({approx}17 nm) were obtained by condensation of 3-glycidoxypropyltriethoxysilane (GPTS) and the surface silanol groups of mesoporous cellular foams (MCFs) and used as the support for immobilization of penicillin G acylase (PGA). The structural properties of G-MCF were characterized by FT-IR, N{sub 2} adsorption, TG-DTA and {sup 29}Si MAS NMR. The studies indicated that the glycidoxypropyl groups were chemically bonded to the silicon atoms on the surface of MCF. The epoxy-functionalized mesoporous cellular foams can provide the microenvironments suitable for the immobilization of PGA, and the enzyme molecules could be immobilized covalently onto the G-MCF under mild conditions by reaction between the amino groups of the enzyme molecules and the epoxy groups on the surface of G-MCF. The PGA immobilized on G-MCF (PGA/G-MCF) exhibited the apparent activity of 1782 IU/g and 46.6% of activity recovery for hydrolyzing penicillin G potassium to produce 6-aminopenicillanic acid at 37 {sup o}C which were higher than that of PGA on pure silica MCF (1521 IU/g and 39.8%, respectively). The kinetic study also indicated that PGA immobilized on G-MCF has a K{sub m} of 2.1 x 10{sup -2} mol/L lower than that of PGA immobilized on the pure silica MCF (5.0 x 10{sup -2} mol/L). These may be attributed to the enhanced surface affinity between G-MCF support and the substrate molecules. Due to the covalent immobilization of PGA molecules on the surface of G-MCF, the immobilized PGA with considerable operational stability was achieved. The activity of PGA/G-MCF is still about 91.4% of its initial activity at the 10th cycle reuse while that of PGA/MCF only remains 41.5% of its initial activity at the same reuse numbers. In addition, the investigation results show the thermal stability and durability on acid or basic medium of PGA immobilized on G

COVALENT IMMOBILIZATION OF INVERTASE ON EPOXY-ACTIVATED POLYANILINE FILMS

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Loredana Vacareanu

2013-08-01

Full Text Available The growing interest in manufacturing and use of biosensors is their rapid and selective detection of the target analyte. The immobilization of the enzymes, onto the appropriate matrix is the key-step in the construction of biosensing devices, considerably affecting its performance. In this study, new polyaniline bearing epoxy groups was synthesized by electrochemical polymerization reactions, as adherent, green film deposited on electrode surface, and was further used as immobilization matrix for invertase enzyme. The immobilization was carried out by condensation reactions between the amino groups of the enzyme molecules and the epoxy groups of polyaniline film. The covalent attachment was achieved by simple immersing the epoxy-activated polyaniline in acetate buffer solution (10 mM, pH 6.0 containing 2mg/mL invertase, for 24 h at 4 ºC, by continuous stirring. The polyaniline films thus obtained were analyzed before and after the invertase attachment, by using FT-IR spectroscopy and SEM microscopy. The presence of the invertase was evaluated by measuring their activity, using UV-Vis spectroscopy, in the presence of a known amount of sucrose as a substrate. These tests, performed for three times under the same conditions, revealed that even after five washes of the polyaniline /invertase electrode to remove the unbounded enzyme, the enzyme remain attached on the polyaniline film, being able to hydrolyze the sucrose presented in the assay solutions.

Polyacrolein/mesoporous silica nanocomposite: Synthesis, thermal stability and covalent lipase immobilization

Energy Technology Data Exchange (ETDEWEB)

Motevalizadeh, Seyed Farshad; Khoobi, Mehdi; Shabanian, Meisam [Department of Medicinal Chemistry, Faculty of Pharmacy and Pharmaceutical Sciences Research Center, Tehran University of Medical Sciences, Tehran 14176 (Iran, Islamic Republic of); Asadgol, Zahra; Faramarzi, Mohammad Ali [Department of Pharmaceutical Biotechnology, Faculty of Pharmacy and Biotechnology Research Center, Tehran University of Medical Sciences, P.O. Box 14155-6451, Tehran 14176 (Iran, Islamic Republic of); Shafiee, Abbas, E-mail: ashafiee@ams.ac.ir [Department of Medicinal Chemistry, Faculty of Pharmacy and Pharmaceutical Sciences Research Center, Tehran University of Medical Sciences, Tehran 14176 (Iran, Islamic Republic of); Center of Excellence in Biothermodynamics, University of Tehran, Tehran (Iran, Islamic Republic of)

2013-12-16

In this work, new polyacrolein/MCM-41 nanocomposites with good phase mixing behavior were prepared through an emulsion polymerization technique. Mesoporous silica was synthesized by in situ assembly of tetraethyl orthosilicate (TEOS) and cetyl trimethyl ammonium bromide (CTAB). The structure and properties of polyacrolein containing nanosized MCM-41 particle (5 and 10 wt%), were investigated by Fourier transform infrared spectroscopy (FTIR), X-ray diffraction, Dynamic light scattering (DLS), scanning electron microscopy (SEM), transmission electron microscopy (TEM), N{sub 2} adsorption techniques, and thermogravimetric (TGA) analyses. The SEM images from the final powder have revealed good dispersion of the MCM-41 nanoparticles throughout polymeric matrix with no distinct voids between two phases. The results indicated that the thermal properties of the nanocomposite were enhanced by addition of MCM-41. Thermomyces lanuginosa lipase (TLL) was used as a model biocatalyst and successfully immobilized with polyacrolein and the nanocomposite via covalent bonds with the aldehyde groups. The activity between free enzyme, polyacrolein, and MCM-41 nanocomposite (10 wt%)-immobilized TLL was compared. The immobilized lipase with the nanocomposite shows better operational stability such as pH tolerance, thermal and storage stability. In addition, the immobilized lipase with the nanocomposite can be easily recovered and retained at 74% of its initial activity after 15 time reuses. - Graphical abstract: The influence of incorporation of mesoporous MCM-41 nanoparticle with polyacrolein on the thermal properties and enzyme immobilization was investigated. - Highlights: • Polyacrolein/MCM-41 nanocomposites were prepared by emulsion polymerization method. • Thermal stability and char residues in nanocomposites were improved. • Nanocomposites significant effects on immobilization of lipase.

Covalent Organic

DEFF Research Database (Denmark)

Vutti, Surendra

chemistry of silicon, InAs and GaAs materials, covalentsurface functionalization using organosilanes, liquid-phase, and vapor-phasefunctionalizations, diazo-transfer reaction, CuAAC click chemistry, different types ofbiorthogonal chemistries, SPAAC chemistry, and cellular interactions of chemically...... immobilization of D-amino acid adhesion peptideson azide functionalized silicon, GaAs and InAs materials by using CuAAC-click chemistry.The covalent immobilization of penetration peptide (TAT) on gold nanotips of InAs NWs isalso demonstrated.In chapter four, the covalent immobilization of GFP on silicon wafers......, GaAs wafers andGaAs NWs is demonstrated. Series of Fmoc-Pra-OH, NHS-PEG5-NHS and BCN-NHSfunctionalized silicon surfaces has been prepared, whereby GFP-N3 and GFP-bicyclononyneare immobilized by using CuAAC and SPAAC chemistry. The specific and covalentimmobilization of GFP-N3 on bicyclononyne...

Covalent immobilization of rabbit-antiaflatoxin-antibodies onto the poly-acrylamideacrylonitrile as well as hybrid material UREASIL and developing an optical immunosensor

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Slavova M.

2017-03-01

Full Text Available The aim of this work is to describe a covalent immobilization of antibodies onto the poly- acrylamide-acrylonitrile or hybrid material UREASIL and creation of optical immunosensor for determination of aflatoxin Bl. For this purpose, mouse-anti-aflatoxin B1 antibodies with oxidized carbohydrate moieties were covalently immobilized on the membranes of polyacrylamide- polyacrylonitrile copolymer, as well as the hybrid material UREASIL. To determine the affinity> binding of the immobilized antibody with afatoxin Bl was used "sandwich" method. Associated with the immobilized antibody sought ingredients interact with a surplus of secondary’ signal antibodies. The described method has been developed as a model system, which can easily be applied for the determination of aflatoxins in samples of different origin. To the best of our knowledge, this is the first study to show that in the establishment of biosensor was used hybrid material UREASIL.

Penicillium subrubescens, a new species efficiently producing inulinase

DEFF Research Database (Denmark)

Mansouri, S.; Houbraken, J.; Samson, R.A.

2013-01-01

Inulin is a reserve carbohydrate in about 15 % of the flowering plants and is accumulated in underground tubers of e.g. chicory, dahlia and Jerusalem artichoke. This carbohydrate consists of linear chains of β-(2,1)-linked fructose attached to a sucrose molecule. Inulinases hydrolyse inulin...... into fructose and glucose. To find efficient inulin degrading fungi, 126 fungal strains from the Fungal Biotechnology Culture Collection (FBCC) at University of Helsinki and 74 freshly isolated strains from soil around Jerusalem artichoke tubers were screened in liquid cultures with inulin as a sole source...... of carbon or ground Jerusalem artichoke tubers, which contains up to 19 % (fresh weight) inulin. Inulinase and invertase activities were assayed by the dinitrosalicylic acid (DNS) method and a freshly isolated Penicillium strain originating from agricultural soil (FBCC 1632) was the most efficient inulinase...

Purification and Characterization of Exo-Inulinase from Paenibacillus sp. d9 Strain.

Science.gov (United States)

Jeza, S; Maseko, S B; Lin, J

2018-02-01

This study intended to purify and characterise exo-inulinase of diesel-degrading Paenibacillus sp. D9. The whole genome sequencing of Paenibacillus sp. D9 revealed to possess the sacC gene that is encoded as exo-inulinase/levanase. This isolate was capable of producing a maximum of 50.9 IU/mL of exo-inulinase activity within 3 days at 30 °C, 200 rpm and pH of 7.0 on minimal salt medium agar supplemented with 1% (w/v) inulin. An exo-inulinase of 58.5 kDa was purified using ammonium sulphate precipitation, HiTrap QFF column and MMC column chromatographies with a specific activity of 4333 IU/mg, 7.1% recovery and a 4.3-fold increase in purity. The purified D9 exo-inulinase had temperature and pH optimum at 40 °C and pH 4.0, respectively, with the Michaelis constant of 5.5 mM and a maximal velocity of 476.2 IU/mg, respectively. Catalytic constant, k cat was calculated to be 42.6 s -1 with a catalytic efficiency (k cat /K m ) of 7.6 s -1  mM -1 . The presence of Ca 2+ enhanced the activity of D9 exo-inulinase while Hg 2+ completely inhibited the activity, other compounds such as Fe 3+ and Cu 2+ had an inhibitory effect. The results of amino acid alignment and the complete degradation of inulin into fructose by the purified enzyme confirmed that inulinase from Paenibacillus sp. D9 is an exo-form. The phylogenetic tree based on the protein sequences indicates that bacterial exo-inulinases possess a common ancestry.

Immobilization of Candida antarctica Lipase B by Covalent Attachment to Green Coconut Fiber

Science.gov (United States)

Brígida, Ana I. S.; Pinheiro, Álvaro D. T.; Ferreira, Andrea L. O.; Pinto, Gustavo A. S.; Gonçalves, Luciana R. B.

The objective of this study was to covalently immobilize Candida antarctica type B lipase (CALB) onto silanized green coconut fibers. Variables known to control the number of bonds between enzyme and support were evaluated including contact time, pH, and final reduction with sodium borohydride. Optimal conditions for lipase immobilization were found to be 2h incubation at both pH 7.0 and 10.0. Thermal stability studies at 60°C showed that the immobilized lipase prepared at pH 10.0 (CALB-10) was 363-fold more stable than the soluble enzyme and 5.4-fold more stable than the biocatalyst prepared at pH 7.0 (CALB-7). CALB-7 was found to have higher specific activity and better stability when stored at 5°C. When sodium borohydride was used as reducing agent on CALB-10 there were no improvement in storage stability and at 60°C stability was reduced for both CALB-7 and CALB-10.

Enhancing inulinase yield by irradiation mutation associated with optimization of culture conditions

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Yafeng Gou

2015-09-01

Full Text Available A new inulinase-producing strain was isolated from rhizosphere soils of Jerusalem artichoke collected from Shihezi (Xinjiang, China using Jerusalem artichoke power (JAP as sole carbon source. It was identified as an Aspergillus niger strain by analysis of 16S rRNA. To improve inulinase production, this fungus was subjected to mutagenesis induced by 60Co γ-irradiation. A genetically stable mutant (designated E12 was obtained and it showed 2.7-fold higher inulinase activity (128 U/mL than the parental strain in the supernatant of a submerged culture. Sequential methodology was used to optimize the inulinase production of stain E12. A screening trial was first performed using Plackett-Burman design and variables with statistically significant effects on inulinase bio-production were identified. These significant factors were further optimized by central composite design experiments and response surface methodology. Finally, it was found that the maximum inulinase production (185 U/mL could be achieved under the optimized conditions namely pH 7.0, yeast extract concentration of 5.0 g/L, JAP concentration of 66.5 g/L, peptone concentration of 29.1 g/L, solution volume of 49.4 mL in 250-mL shake flasks, agitation speed of 180 rpm, and fermentation time of 60 h. The yield of inulinase under optimized culture conditions was approximately 1.4-fold of that obtained by using basal culture medium. These findings are of significance for the potential industrial application of the mutant E12.

Site-Specific, Covalent Immobilization of Dehalogenase ST2570 Catalyzed by Formylglycine-Generating Enzymes and Its Application in Batch and Semi-Continuous Flow Reactors

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Hui Jian

2016-07-01

Full Text Available Formylglycine-generating enzymes can selectively recognize and oxidize cysteine residues within the sulfatase sub motif at the terminus of proteins to form aldehyde-bearing formylglycine (FGly residues, and are normally used in protein labeling. In this study, an aldehyde tag was introduced to proteins using formylglycine-generating enzymes encoded by a reconstructed set of the pET28a plasmid system for enzyme immobilization. The haloacid dehalogenase ST2570 from Sulfolobus tokodaii was used as a model enzyme. The C-terminal aldehyde-tagged ST2570 (ST2570CQ exhibited significant enzymological properties, such as new free aldehyde groups, a high level of protein expression and improved enzyme activity. SBA-15 has widely been used as an immobilization support for its large surface and excellent thermal and chemical stability. It was functionalized with amino groups by aminopropyltriethoxysilane. The C-terminal aldehyde-tagged ST2570 was immobilized to SBA-15 by covalent binding. The site-specific immobilization of ST2570 avoided the chemical denaturation that occurs in general covalent immobilization and resulted in better fastening compared to physical adsorption. The site-specific immobilized ST2570 showed 3-fold higher thermal stability, 1.2-fold higher catalytic ability and improved operational stability than free ST2570. The site-specific immobilized ST2570 retained 60% of its original activity after seven cycles of batch operation, and it was superior to the ST2570 immobilized to SBA-15 by physical adsorption, which loses 40% of its original activity when used for the second time. It is remarkable that the site-specific immobilized ST2570 still retained 100% of its original activity after 10 cycles of reuse in the semi-continuous flow reactor. Overall, these results provide support for the industrial-scale production and application of site-specific, covalently immobilized ST2570.

Enhanced production of extracellular inulinase by the yeast Kluyveromyces marxianus in xylose catabolic state.

Science.gov (United States)

Hoshida, Hisashi; Kidera, Kenta; Takishita, Ryuta; Fujioka, Nobuhisa; Fukagawa, Taiki; Akada, Rinji

2018-06-01

The production of extracellular proteins by the thermotolerant yeast Kluyveromyces marxianus, which utilizes various sugars, was investigated using media containing sugars such as glucose, galactose, and xylose. SDS-PAGE analysis of culture supernatants revealed abundant production of an extracellular protein when cells were grown in xylose medium. The N-terminal sequence of the extracellular protein was identical to a part of the inulinase encoded by INU1 in the genome. Inulinase is an enzyme hydrolyzing β-2,1-fructosyl bond in inulin and sucrose and is not required for xylose assimilation. Disruption of INU1 in the strain DMKU 3-1042 lost the production of the extracellular protein and resulted in growth defect in sucrose and inulin media, indicating that the extracellular protein was inulinase (sucrase). In addition, six K. marxianus strains among the 16 strains that were analyzed produced more inulinase in xylose medium than in glucose medium. However, expression analysis indicated that the INU1 promoter activity was lower in the xylose medium than in the glucose medium, suggesting that enhanced production of inulinase is controlled in a post-transcriptional manner. The production of inulinase was also higher in cultures with more agitation, suggesting that oxygen supply affects the production of inulinase. Taken together, these results suggest that both xylose and oxygen supply shift cellular metabolism to enhance the production of extracellular inulinase. Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

OPTIMIZATION OF INULINASE PRODUCTION USING COPRA WASTE BY Kluyveromyces marxianus var. marxianus

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M. DILIPKUMAR

2010-12-01

Full Text Available Kluyveromyces marxianus var. marxianus was found to secrete a large amount of extracellular inulinase in to the medium. The optimization of inulinase pro¬duction using copra waste as a carbon source was performed with statistical methodology based on experimental designs. The screening of eighteen nut¬rients for their influence on inulinase production was achieved using a Plackett––Burman design. Corn steep liquor, (NH42SO4, ZnSO47H2O, K2HPO4 and urea were selected based on their positive influence on inulinase production. The selected components were optimized using response surface methodology (RSM. The optimum conditions are: corn steep liquor – 0.0560 (g/gds, (NH42SO4 – 0.0084 (g/gds, ZnSO47H2O – 0.0254 (g/gds, K2HPO4 – 0.0037 (g/gds and urea - 0.02147 (g/gds. These conditions were validated experimentally which revealed an enhanced inulinase yield of 372 U/gds.

Covalent immobilization of porcine pancreatic lipase on carboxyl-activated magnetic nanoparticles: Characterization and application for enzymatic inhibition assays

International Nuclear Information System (INIS)

Zhu, Yuan-Ting; Ren, Xiao-Yun; Liu, Yi-Ming; Wei, Ying; Qing, Lin-Sen; Liao, Xun

2014-01-01

Using carboxyl functionalized silica-coated magnetic nanoparticles (MNPs) as carrier, a novel immobilized porcine pancreatic lipase (PPL) was prepared through the 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride/N-hydroxysuccinimide (EDC/NHS) coupling reaction. Transmission electron microscopic images showed that the synthesized nanoparticles (Fe 3 O 4 –SiO 2 ) possessed three dimensional core–shell structures with an average diameter of ∼ 20 nm. The effective enzyme immobilization onto the nanocomposite was confirmed by atomic force microscopic (AFM) analysis. Results from Fourier-transform infrared spectroscopy (FT-IR), Bradford protein assay, and thermo-gravimetric analysis (TGA) indicated that PPL was covalently attached to the surface of magnetic nanoparticles with a PPL immobilization yield of 50 mg enzyme/g MNPs. Vibrating sample magnetometer (VSM) analysis revealed that the MNPs-PPL nanocomposite had a high saturation magnetization of 42.25 emu·g −1 . The properties of the immobilized PPL were investigated in comparison with the free enzyme counterpart. Enzymatic activity, reusability, thermo-stability, and storage stability of the immobilized PPL were found significantly superior to those of the free one. The K m and the V max values (0.02 mM, 6.40 U·mg −1 enzyme) indicated the enhanced activity of the immobilized PPL compared to those of the free enzyme (0.29 mM, 3.16 U·mg −1 enzyme). Furthermore, at an elevated temperature of 70 °C, immobilized PPL retained 60% of its initial activity. The PPL-MNPs nanocomposite was applied in the enzyme inhibition assays using orlistat, and two natural products isolated from oolong tea (i.e., EGCG and EGC) as the test compounds. - Highlights: • Porcine pancreatic lipase was firstly covalently immobilized onto carboxylated MNPs. • Immobilized porcine pancreatic lipase (PPL) was characterized by various techniques. • MNPs-PPL showed higher activity, reusability, and thermo-stability than

Covalent immobilization of porcine pancreatic lipase on carboxyl-activated magnetic nanoparticles: Characterization and application for enzymatic inhibition assays

Energy Technology Data Exchange (ETDEWEB)

Zhu, Yuan-Ting [Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041 (China); University of Chinese Academy of Sciences, Beijing 100049 (China); Ren, Xiao-Yun [Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041 (China); Liu, Yi-Ming [Department of Chemistry and Biochemistry, Jackson State University, 1400 Lynch St., Jackson, MS 39217 (United States); Wei, Ying [Changzhi Medical College, Changzhi 046000 (China); Qing, Lin-Sen [Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041 (China); Liao, Xun, E-mail: liaoxun@cib.ac.cn [Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041 (China)

2014-05-01

Using carboxyl functionalized silica-coated magnetic nanoparticles (MNPs) as carrier, a novel immobilized porcine pancreatic lipase (PPL) was prepared through the 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride/N-hydroxysuccinimide (EDC/NHS) coupling reaction. Transmission electron microscopic images showed that the synthesized nanoparticles (Fe{sub 3}O{sub 4}–SiO{sub 2}) possessed three dimensional core–shell structures with an average diameter of ∼ 20 nm. The effective enzyme immobilization onto the nanocomposite was confirmed by atomic force microscopic (AFM) analysis. Results from Fourier-transform infrared spectroscopy (FT-IR), Bradford protein assay, and thermo-gravimetric analysis (TGA) indicated that PPL was covalently attached to the surface of magnetic nanoparticles with a PPL immobilization yield of 50 mg enzyme/g MNPs. Vibrating sample magnetometer (VSM) analysis revealed that the MNPs-PPL nanocomposite had a high saturation magnetization of 42.25 emu·g{sup −1}. The properties of the immobilized PPL were investigated in comparison with the free enzyme counterpart. Enzymatic activity, reusability, thermo-stability, and storage stability of the immobilized PPL were found significantly superior to those of the free one. The K{sub m} and the V{sub max} values (0.02 mM, 6.40 U·mg{sup −1} enzyme) indicated the enhanced activity of the immobilized PPL compared to those of the free enzyme (0.29 mM, 3.16 U·mg{sup −1} enzyme). Furthermore, at an elevated temperature of 70 °C, immobilized PPL retained 60% of its initial activity. The PPL-MNPs nanocomposite was applied in the enzyme inhibition assays using orlistat, and two natural products isolated from oolong tea (i.e., EGCG and EGC) as the test compounds. - Highlights: • Porcine pancreatic lipase was firstly covalently immobilized onto carboxylated MNPs. • Immobilized porcine pancreatic lipase (PPL) was characterized by various techniques. • MNPs-PPL showed higher activity

Covalent Immobilization of Peroxidase onto Hybrid Membranes for the Construction of Optical Biosensor

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Lyubov Yotova

2015-06-01

Full Text Available The aim of this study is to covalently immobilize horse radish peroxidase (HRP onto new hybrid membranes synthesized by the sol-gel method based on silica precursors, dendrimers and cellulose derivatives. This new system will be used for designing biosensor. For investigation of the properties of membranes, HRP was used as a modeling enzyme. Kinetic parameters, pH and temperature optimum were determined, and the structure of the membranes surface was examined. Results showed higher relative and residual activity of HRP immobilized onto membranes with cellulose acetate butyrate with high molecular weight CAB/H. This novel biosensor could offer a simple, cheap and rapid tool with enhanced sensing performance as well as having potentials to find application in medicine, pharmacy, food and process control and environmental monitoring.

Covalent immobilization of lysozyme onto woven and knitted crimped polyethylene terephthalate grafts to minimize the adhesion of broad spectrum pathogens

International Nuclear Information System (INIS)

Al Meslmani, Bassam M.; Mahmoud, Gihan F.; Leichtweiß, Thomas; Strehlow, Boris; Sommer, Frank O.; Lohoff, Michael D.; Bakowsky, Udo

2016-01-01

Graft-associated infections entirely determine the short-term patency of polyethylene terephthalate PET cardiovascular graft. We attempted to enzymatically inhibit the initial bacterial adhesion to PET grafts using lysozyme. Lysozyme was covalently immobilized onto woven and knitted forms of crimped PET grafts by the end-point method. Our figures of merit revealed lysozyme immobilization yield of 15.7 μg/cm"2, as determined by the Bradford assay. The activity of immobilized lysozyme on woven and knitted PET manifested 58.4% and 55.87% using Micrococcus lysodeikticus cells, respectively. Noteworthy, the adhesion of vein catheter-isolated Staphylococcus epidermidis decreased by 6- to 8-folds and of Staphylococcus aureus by 11- to 12-folds, while the Gram-negative Escherichia coli showed only a decrease by 3- to 4-folds. The anti-adhesion efficiency was specific for bacterial cells and no significant effect was observed on adhesion and growth of L929 cells. In conclusion, immobilization of lysozyme onto PET grafts can inhibit the graft-associated infection. - Highlights: • Lysozyme was covalently immobilized on crimped polyethylene terephthalate (PET). • The activity of immobilized lysozyme was meaningfully reduced. • The maintained activity significantly declined the adhesion of Gram-positive stains. • The enzymatic anti-adhesion efficiency reported lesser extent against Gram-negative. • The anti-bacterial activity displayed no significant effect on cells compatibility.

Covalent immobilization of lysozyme onto woven and knitted crimped polyethylene terephthalate grafts to minimize the adhesion of broad spectrum pathogens

Energy Technology Data Exchange (ETDEWEB)

Al Meslmani, Bassam M., E-mail: almeslmanib@yahoo.com [Department of Pharmaceutical Technology and Biopharmaceutics, Marburg University, Ketzerbach 63, 35037 Marburg (Germany); Mahmoud, Gihan F., E-mail: mahmoudg@staff.uni-marburg.de [Department of Pharmaceutical Technology and Biopharmaceutics, Marburg University, Ketzerbach 63, 35037 Marburg (Germany); Department of Pharmaceutics and Industrial Pharmacy, Helwan University, Ain Helwan, 11795 Cairo (Egypt); Leichtweiß, Thomas, E-mail: Thomas.Leichtweiss@phys.Chemie.uni-giessen.de [Institute of Physical Chemistry, Justus-Liebig-University Giessen, Heinrich-Buff-Ring 58, 35392 Giessen (Germany); Strehlow, Boris, E-mail: strehlo4@staff.uni-marburg.de [Department of Pharmaceutical Technology and Biopharmaceutics, Marburg University, Ketzerbach 63, 35037 Marburg (Germany); Sommer, Frank O., E-mail: sommerf@med.uni-marburg.de [Institute for Medical Microbiology and Hospital Hygiene, Marburg University, Hans Meerwein Str 2, 35032 Marburg (Germany); Lohoff, Michael D., E-mail: lohoff@med.uni-marburg.de [Institute for Medical Microbiology and Hospital Hygiene, Marburg University, Hans Meerwein Str 2, 35032 Marburg (Germany); Bakowsky, Udo, E-mail: ubakowsky@aol.com [Department of Pharmaceutical Technology and Biopharmaceutics, Marburg University, Ketzerbach 63, 35037 Marburg (Germany)

2016-01-01

Graft-associated infections entirely determine the short-term patency of polyethylene terephthalate PET cardiovascular graft. We attempted to enzymatically inhibit the initial bacterial adhesion to PET grafts using lysozyme. Lysozyme was covalently immobilized onto woven and knitted forms of crimped PET grafts by the end-point method. Our figures of merit revealed lysozyme immobilization yield of 15.7 μg/cm{sup 2}, as determined by the Bradford assay. The activity of immobilized lysozyme on woven and knitted PET manifested 58.4% and 55.87% using Micrococcus lysodeikticus cells, respectively. Noteworthy, the adhesion of vein catheter-isolated Staphylococcus epidermidis decreased by 6- to 8-folds and of Staphylococcus aureus by 11- to 12-folds, while the Gram-negative Escherichia coli showed only a decrease by 3- to 4-folds. The anti-adhesion efficiency was specific for bacterial cells and no significant effect was observed on adhesion and growth of L929 cells. In conclusion, immobilization of lysozyme onto PET grafts can inhibit the graft-associated infection. - Highlights: • Lysozyme was covalently immobilized on crimped polyethylene terephthalate (PET). • The activity of immobilized lysozyme was meaningfully reduced. • The maintained activity significantly declined the adhesion of Gram-positive stains. • The enzymatic anti-adhesion efficiency reported lesser extent against Gram-negative. • The anti-bacterial activity displayed no significant effect on cells compatibility.

Surface functionalization of chitosan-coated magnetic nanoparticles for covalent immobilization of yeast alcohol dehydrogenase from Saccharomyces cerevisiae

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Li, Gui-yin; Zhou, Zhi-de; Li, Yuan-jian; Huang, Ke-long; Zhong, Ming

2010-12-01

A novel and efficient immobilization of yeast alcohol dehydrogenase (YADH, EC1.1.1.1) from Saccharomyces cerevisiae has been developed by using the surface functionalization of chitosan-coated magnetic nanoparticles (Fe 3O 4/KCTS) as support. The magnetic Fe 3O 4/KCTS nanoparticles were prepared by binding chitosan alpha-ketoglutaric acid (KCTS) onto the surface of magnetic Fe 3O 4 nanoparticles. Later, covalent immobilization of YADH was attempted onto the Fe 3O 4/KCTS nanoparticles. The effect of various preparation conditions on the immobilized YADH process such as immobilization time, enzyme concentration and pH was investigated. The influence of pH and temperature on the activity of the free and immobilized YADH using phenylglyoxylic acid as substrate has also been studied. The optimum reaction temperature and pH value for the enzymatic conversion catalyzed by the immobilized YADH were 30 °C and 7.4, respectively. Compared to the free enzyme, the immobilized YADH retained 65% of its original activity and exhibited significant thermal stability and good durability.

Effects of carbon sources, oxygenation and ethanol on the production of inulinase by Kluyveromyces marxianus YX01

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JIAOQI GAO

2012-01-01

Full Text Available Inulinase is one of the most important factors in consolidated bioprocessing, which combines enzyme production, inulin saccharification, and ethanol fermentation into a single process. In our study, inulinase production and cell growth of Kluyveromyces marxianus YX01 under different conditions were studied. Carbon source was shown to be significant on the production of inulinase, because the activity of inulinase was higher using inulin as a carbon source compared with glucose or fructose. The concentration of the carbon source had a repressive effect on the activity of inulinase. When the concentration was increased to 60 g/L, inulinase activity was only 50% compared with carbon source concentration of 20 g/L. Enzyme activity was also strongly influenced by aeration rate. It has been shown that the activity of inulinase and cell growth under anaerobic conditions were maintained at low levels, but aeration at 1.0 vvm (air volume/broth volume minute led to higher activity. Inulinase activity per unit biomass was not significantly different under different aeration rates. Ethanol had a repressive effect on the cell growth. Cells ceased growing when the level of ethanol was greater than 9% (v/v, but ethanol did not affect the activity of secreted inulinase and the enzyme was stable at ethanol concentration up to 15%.

Improving Properties of a Novel β-Galactosidase from Lactobacillus plantarum by Covalent Immobilization

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Rocio Benavente

2015-04-01

Full Text Available A novel β-galactosidase from Lactobacillus plantarum (LPG was over-expressed in E. coli and purified via a single chromatographic step by using lowly activated IMAC (immobilized metal for affinity chromatography supports. The pure enzyme exhibited a high hydrolytic activity of 491 IU/mL towards o-nitrophenyl β-d-galactopyranoside. This value was conserved in the presence of different divalent cations and was quite resistant to the inhibition effects of different carbohydrates. The pure multimeric enzyme was stabilized by multipoint and multisubunit covalent attachment on glyoxyl-agarose. The glyoxyl-LPG immobilized preparation was over 20-fold more stable than the soluble enzyme or the one-point CNBr-LPG immobilized preparation at 50 °C. This β-galactosidase was successfully used in the hydrolysis of lactose and lactulose and formation of different oligosaccharides was detected. High production of galacto-oligosaccharides (35% and oligosaccharides derived from lactulose (30% was found and, for the first time, a new oligosaccharide derived from lactulose, tentatively identified as 3'-galactosyl lactulose, has been described.

Strategies to balance covalent and non-covalent biomolecule attachment within collagen-GAG biomaterials.

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Pence, Jacquelyn C; Gonnerman, Emily A; Bailey, Ryan C; Harley, Brendan A C

2014-09-01

Strategies to integrate instructive biomolecular signals into a biomaterial are becoming increasingly complex and bioinspired. While a large majority of reports still use repeated treatments with soluble factors, this approach can be prohibitively costly and difficult to translate in vivo for applications where spatial control over signal presentation is necessary. Recent efforts have explored the use of covalent immobilization of biomolecules to the biomaterial, via both bulk (ubiquitous) as well as spatially-selective light-based crosslinking, as a means to both enhance stability and bioactivity. However, little is known about how processing conditions during immobilization impact the degree of unintended non-covalent interactions, or fouling, that takes place between the biomaterial and the biomolecule of interest. Here we demonstrate the impact of processing conditions for bulk carbodiimide (EDC) and photolithography-based benzophenone (BP) crosslinking on specific attachment vs. fouling of a model protein (Concanavalin A, ConA) within collagen-glycosaminoglycan (CG) scaffolds. Collagen source significantly impacts the selectivity of biomolecule immobilization. EDC crosslinking intensity and ligand concentration significantly impacted selective immobilization. For benzophenone photoimmobilization we observed that increased UV exposure time leads to increased ConA immobilization. Immobilization efficiency for both EDC and BP strategies was maximal at physiological pH. Increasing ligand concentration during immobilization process led to enhanced immobilization for EDC chemistry, no impact on BP immobilization, but significant increases in non-specific fouling. Given recent efforts to covalently immobilize biomolecules to a biomaterial surface to enhance bioactivity, improved understanding of the impact of crosslinking conditions on selective attachment versus non-specific fouling will inform the design of instructive biomaterials for applications across tissue

Bioelectrochemistry of non-covalent immobilized alcohol dehydrogenase on oxidized diamond nanoparticles.

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Nicolau, Eduardo; Méndez, Jessica; Fonseca, José J; Griebenow, Kai; Cabrera, Carlos R

2012-06-01

Diamond nanoparticles are considered a biocompatible material mainly due to their non-cytotoxicity and remarkable cellular uptake. Model proteins such as cytochrome c and lysozyme have been physically adsorbed onto diamond nanoparticles, proving it to be a suitable surface for high protein loading. Herein, we explore the non-covalent immobilization of the redox enzyme alcohol dehydrogenase (ADH) from Saccharomyces cerevisiae (E.C.1.1.1.1) onto oxidized diamond nanoparticles for bioelectrochemical applications. Diamond nanoparticles were first oxidized and physically characterized by X-ray diffraction (XRD), FT-IR and TEM. Langmuir isotherms were constructed to investigate the ADH adsorption onto the diamond nanoparticles as a function of pH. It was found that a higher packing density is achieved at the isoelectric point of the enzyme. Moreover, the relative activity of the immobilized enzyme on diamond nanoparticles was addressed under optimum pH conditions able to retain up to 70% of its initial activity. Thereafter, an ethanol bioelectrochemical cell was constructed by employing the immobilized alcohol dehydrogenase onto diamond nanoparticles, this being able to provide a current increment of 72% when compared to the blank solution. The results of this investigation suggest that this technology may be useful for the construction of alcohol biosensors or biofuel cells in the near future. Copyright © 2011 Elsevier B.V. All rights reserved.

Optimization of pectinase immobilization on grafted alginate-agar gel beads by 24 full factorial CCD and thermodynamic profiling for evaluating of operational covalent immobilization.

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Abdel Wahab, Walaa A; Karam, Eman A; Hassan, Mohamed E; Kansoh, Amany L; Esawy, Mona A; Awad, Ghada E A

2018-07-01

Pectinase produced by a honey derived from the fungus Aspergillus awamori KX943614 was covalently immobilized onto gel beads made of alginate and agar. Polyethyleneimine, glutaraldehyde, loading time and enzyme's units were optimized by 2 4 full factorial central composite design (CCD). The immobilization process increased the optimal working pH for the free pectinase from 5 to a broader range of pH4.5-5.5 and the optimum operational temperature from 55°C to a higher temperature, of 60°C, which is favored to reduce the enzyme's microbial contamination. The thermodynamics studies showed a thermal stability enhancement against high temperature for the immobilized formula. Moreover, an increase in half-lives and D-values was achieved. The thermodynamic studies proved that immobilization of pectinase made a remarkable increase in enthalpy and free energy because of enzyme stability enhancement. The reusability test revealed that 60% of pectinase's original activity was retained after 8 successive cycles. This gel formula may be convenient for immobilization of other industrial enzymes. Copyright © 2018 Elsevier B.V. All rights reserved.

Solid-State Fermentation of Carrot Pomace for the Production of Inulinase by Penicillium oxalicum BGPUP-4.

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Singh, Ram Sarup; Chauhan, Kanika; Singh, Jagroop; Pandey, Ashok; Larroche, Christian

2018-03-01

Inulinases are an important class of industrial enzymes which are used for the production of high-fructose syrup and fructooligosaccharides. Inulin, a polyfructan, is generally employed for the production of inulinase, which is a very expensive substrate. A number of agroindustrial residues have been used for cost-effective production of inulinases. In the present study, carrot pomace was selected as a substrate for the production of inulinase by Penicillium oxalicum BGPUP-4 in solid-state fermentation. Carrot pomace is one of the good substrates for bioprocesses, because it is rich in soluble and insoluble carbohydrates. A central composite rotatable design (CCRD) used in response surface methodology was employed for the optimal production of inulinase from carrot pomace. Using CCRD, 15 runs were practiced to optimize the range of three independent variables: moisture content (70-90%), incubation time (4-6 days) and pH (5.0-7.0) for inulinase production. Carrot pomace supplemented with 0.5% inulin as an inducer, 0.2% NH 4 H 2 PO 4 , 0.2% NaNO 3 , 0.2% KH 2 PO 4 , 0.05% MgSO 4 ·7H 2 O and 0.001% FeSO 4 ·7H 2 O was used for the production of inulinase in solid-state fermentation at 30 °C. Inulinase production (322.10 IU per g of dry substrate) was obtained under the optimized conditions, i.e . moisture content of 90%, incubation time 4 days and pH=7.0. The corresponding inulinase/invertase (I/S) ratio (3.38) was also high, which indicates the inulolytic nature of the enzyme. Multiple correlation coefficients R for inulinase production and I/S ratio were 0.9995 and 0.9947, respectively. The R value very close to one indicates an excellent correlation between experimental and predicted results.

Statistical optimization of process parameters for inulinase production from Tithonia weed by Arthrobacter mysorens strain no.1.

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Kamble, Prajakta P; Kore, Maheshkumar V; Patil, Sushama A; Jadhav, Jyoti P; Attar, Yasmin C

2018-06-01

Tithonia rotundifolia is an easily available and abundant inulin rich weed reported to be competitive and allelopathic. This weed inulin is hydrolyzed by inulinase into fructose. Response surface methodology was employed to optimize culture conditions for the inulinase production from Arthrobacter mysorens strain no.1 isolated from rhizospheric area of Tithonia weed. Initially, Plackett- Burman design was used for screening 11 nutritional parameters for inulinase production including inulin containing weeds as cost effective substrate. The experiment shows that amongst the 11 parameters studied, K 2 HPO 4 , Inulin, Agave sisalana extract and Tithonia rotundifolia were the most significant variables for inulinase production. Quantitative effects of these 4 factors were further investigated using Box Behnken design. The medium having 0.27% K 2 HPO 4 , 2.54% Inulin, 6.57% Agave sisalana extract and 7.27% Tithonia rotundifolia extract were found to be optimum for maximum inulinase production. The optimization strategies used showed 2.12 fold increase in inulinase yield (1669.45 EU/ml) compared to non-optimized medium (787 EU/ml). Fructose produced by the action of inulinase was further confirmed by spectrophotometer, osazone, HPTLC and FTIR methods. Thus Tithonia rotundifolia can be used as an eco-friendly, economically feasible and promising alternative substrate for commercial inulinase production yielding fructose from Arthrobacter mysorens strain no.1. Copyright © 2018 Elsevier B.V. All rights reserved.

[Oligonucleotide derivatives in the nucleic acid hybridization analysis. I. Covalent immobilization of oligonucleotide probes onto the nylon].

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Dmitrienko, E V; Pyshnaia, I A; Pyshnyĭ, D V

2010-01-01

The features of UV-induced immobilization of oligonucleotides on a nylon membranes and the effectiveness of enzymatic labeling of immobilized probes at heterophase detection of nucleic acids are studied. Short terminal oligothymidilate (up to 10 nt) sequences are suggested to attach to the probe via a flexible ethylene glycol based linker. The presence of such fragment enhances the intensity of immobilization and reduces UV-dependent degradation of the targeted (sequence-specific) part of the probe by reducing the dose needed for the immobilization of DNA. The optimum dose of UV-irradiation is determined to be ~0.4 J/cm(2) at the wavelength 254 nm. This dose provides high level of hybridization signal for immobilized probes with various nucleotide composition of the sequence specific moiety. The amide groups of the polyamide are shown to play the key role in the photoinduced immobilization of nucleic acids, whereas the primary amino groups in the structure of PA is not the center responsible for the covalent binding of DNA by UV-irradiation, as previously believed. Various additives in the soaking solution during the membrane of UV-dependent immobilization of probes are shown to influence its effectiveness. The use of alternative to UV-irradiation system of radical generation are shown to provide the immobilization of oligonucleotides onto the nylon membrane.

Application of agave subproducts for production of microbial inulinases

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Huerta Alcocer, S.A.

2014-07-01

Full Text Available Mexico is the center of origin of the genus Agave spp. with 159 endemic species, 75 % of all known species. The main use of these plants is in the production of alcoholic beverages, mainly tequila, which is obtained from the “pines” of A. tequilana Weber var. Azul. During tequila production, a huge amount of waste is produced, including leaves that are not used. Main compounds found in these subproducts are polysaccharides, specifically inulin. Current review focuses on the use of these agricultural wastes to obtain inulinase production, and in the production of high fructose syrup using these enzymes in these wastes. Currently, high fructose syrups, especially oligosaccharides, have prompted much interest for their benefits to health due to its low glycemic index and their function as prebiotic in the intestinal flora. The main microorganisms involved with inulinases production belong to the genera Aspergillus and Kluyveromyces. The latter is preferred in the industry due to its high growth rate, high temperature tolerance (52 °C and by having the GRAS (Generally Recognized As Safe status. This review emphasizes on the advances achieved in the production of inulinases by yeast K. marxianus.

Glucose-free fructose production from Jerusalem artichoke using a recombinant inulinase-secreting Saccharomyces cerevisiae strain.

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Yu, Jing; Jiang, Jiaxi; Ji, Wangming; Li, Yuyang; Liu, Jianping

2011-01-01

Using inulin (polyfructose) obtained from Jerusalen artichokes, we have produced fructose free of residual glucose using a recombinant inulinase-secreting strain of Saccharomyces cerevisiae in a one-step fermentation of Jerusalem artichoke tubers. For producing fructose from inulin, a recombinant inulinase-producing Saccharomyce cerevisiae strain was constructed with a deficiency in fructose uptake by disruption of two hexokinase genes hxk1 and hxk2. The inulinase gene introduced into S. cerevisiae was cloned from Kluyveromyces cicerisporus. Extracellular inulinase activity of the recombinant hxk-mutated S. cerevisiae strain reached 31 U ml(-1) after 96 h growth. When grown in a medium containing Jerusalem artichoke tubers as the sole component without any additives, the recombinant yeast accumulated fructose up to 9.2% (w/v) in the fermentation broth with only 0.1% (w/v) glucose left after 24 h.

Production of inulinase by Xanthomonas campestris pv phaseoli using onion (Allium cepa) and garlic (Allium sativum) peels in solid state cultivation.

Science.gov (United States)

Ayyachamy, M; Khelawan, K; Pillay, D; Permaul, K; Singh, S

2007-10-01

To access inulinase production by Xanthomonas campestris pv phaseoli using the submerged and solid state cultivation (SSC) methods. Various carbon sources, inulin-rich solid substrates and pure synthetic inulin were tested for their efficiency in inulinase induction. The highest inulinase production (17.42 IU ml(-1)) in submerged cultures of X. campestris was observed with inulin as a carbon source with an initial pH, temperature and agitation of 7.0, 37 degrees C and 150 rev min(-1) respectively. Among the various substrates, garlic peels (117 IU gds(-1)) and onion peels (101 IU gds(-1)) were found to be the best for inulinase production. The inulinase production level of X. campestris was 6.7-fold higher in garlic and 5.8-fold in onion, under optimized SSC conditions compared with the submerged culture. This is the first report on inulinase production from garlic and onion peels by X. campestris using SSC. SSC is an efficient method for inulinase production by X. campestris for commercial applications.

Surface functionalization of polyethylene via covalent immobilization of O-stearoyl-chitosan

International Nuclear Information System (INIS)

Xin, Zhirong; Hou, Juan; Ding, Jiaotong; Yang, Zongfeng; Yan, Shunjie; Liu, Chan

2013-01-01

When used in blood-contacting field, the hemocompatibility of polyethylene (PE) needs further to be improved. In this article, O-stearoyl-chitosans (OSC) with different esterification degrees were successfully prepared via changing the ratios of chitosan and stearoyl chloride for decreasing the cationic and hydrogen bond strength, thus improving the solubility of chitosan. The PE film was grafted with carboxyl groups of acrylic acid (AA) (PE-g-PAA) by means of O 2 plasma pre-treatment and UV-induced graft polymerization, and then PE-g-PAA was used for covalent immobilization of OSC. The above surface modification was confirmed by ATR-FTIR and XPS. Effect of the UV-irradiated graft polymerization parameters, i.e., the discharge power, the plasma pretreatment time, the UV irradiation time and the monomer concentration on the grafting density of AA was investigated. Relative to the value of about 107° for the virgin sample, the water contact angle (WCA) of the PAA-grafted film was about 50°. After the further immobilization of OSC onto the PAA-grafted film, the strength of negative charge of the PAA-grafted surface was decreased by the electropositive OSC, thus presenting a WCA value of about 62° and the excellent performance of anti-platelet adhesion with respect to the virgin and PAA-grafted samples.

Comparative study of two purified inulinases from thermophile Thielavia Terrestris NRRL 8126 and mesophile Aspergillus Foetidus NRRL 337 grown on Cichorium Intybus l

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Eman Mohamed Fawzi

2011-06-01

Full Text Available Thirty fungal species grown on Cichorium intybus L. root extract as a sole carbon source, were screened for the production of exo-inulinase activities. The thermophile Thielavia terrestris NRRL 8126 and mesophile Aspergillus foetidus NRRL 337 gave the highest production levels of inulinases I & II at 50 and 24 ºC respectively. Yeast extract and peptone were the best nitrogen sources for highest production of inulinases I & II at five and seven days of incubation respectively. The two inulinases I & II were purified to homogeneity by gel-filtration and ion-exchange chromatography with 66.0 and 42.0 fold of purification respectively. The optimum temperatures of purified inulinases I & II were 75 and 50 ºC respectively. Inulinase I was more thermostable than the other one. The optimum pH for activity was found to be 4.5 and 5.5 for inulinases I & II respectively. A comparatively lower Michaelis-Menten constant (2.15 mg/ml and higher maximum initial velocity (115 µmol/min/mg of protein for inulinase I on inulin demonstrated the exoinulinase's greater affinity for inulin substrate. These findings are significant for its potential industrial application. The molecular mass of the inulinases I & II were estimated to be 72 & 78 kDa respectively by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Optimisation of Inulinase Production by Kluyveromyces bulgaricus

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Darija Vranešić

2002-01-01

Full Text Available The present work is based on observation of the effects of pH and temperature of fermentation on the production of microbial enzyme inulinase by Kluyveromyces marxianus var. bulgaricus. Inulinase hydrolyzes inulin, a polysaccharide which can be isolated from plants such as Jerusalem artichoke, chicory or dahlia, and transformed into pure fructose or fructooligosaccharides. Fructooligosaccharides have great potential in food industry because they can be used as calorie-reduced compounds and noncariogenic sweeteners as well as soluble fibre and prebiotic compounds. Fructose formation from inulin is a single step enzymatic reaction and yields are up to 95 % the fructose. On the contrary, conventional fructose production from starch needs at least three enzymatic steps, yielding only 45 % of fructose. The process of inulinase production was optimised by using experimental design method. pH value of the cultivation medium showed to be the most significant variable and it should be maintained at optimum value of 3.6. The effect of temperature was slightly lower and optimal values were between 30 and 33 °C. At a low pH value of the cultivation medium, the microorganism was not able to producem enough enzyme and enzyme activities were low. Similar effect was caused by high temperature. The highest values of enzyme activities were achieved at optimal fermentation conditions and the values were: 100.16–124.36 IU/mL (with sucrose as substrate for determination of enzyme activity or 8.6–11.6 IU/mL (with inulin as substrate, respectively. The method of factorial design and response surface analysis makes it possible to study several factors simultaneously, to quantify the individual effect of each factor and to investigate their possible interactions. As a comparison to this method, optimisation of a physiological enzyme activity model depending on pH and temperature was also studied.

Enhanced exo-inulinase activity and stability by fusion of an inulin-binding module.

Science.gov (United States)

Zhou, Shun-Hua; Liu, Yuan; Zhao, Yu-Juan; Chi, Zhe; Chi, Zhen-Ming; Liu, Guang-Lei

2016-09-01

In this study, an inulin-binding module from Bacillus macerans was successfully fused to an exo-inulinase from Kluyveromyces marxianus, creating a hybrid functional enzyme. The recombinant exo-inulinase (rINU), the hybrid enzyme (rINUIBM), and the recombinant inulin-binding module (rIBM) were, respectively, heterologously expressed and biochemically characterized. It was found that both the inulinase activity and the catalytic efficiency (k cat/K m(app)) of the rINUIBM were considerably higher than those of rINU. Though the rINU and the rINUIBM shared the same optimum pH of 4.5, the optimum temperature of the rINUIBM (60 °C) was 5 °C higher than that of the rINU. Notably, the fused IBM significantly enhanced both the pH stability and the thermostability of the rINUIBM, suggesting that the rINUIBM obtained would have more extensive potential applications. Furthermore, the fusion of the IBM could substantially improve the inulin-binding capability of the rINUIBM, which was consistent with the determination of the K m(app). This meant that the fused IBM could play a critical role in the recognition of polysaccharides and enhanced the hydrolase activity of the associated inulinase by increasing enzyme-substrate proximity. Besides, the extra supplement of the independent non-catalytic rIBM could also improve the inulinase activity of the rINU. However, this improvement was much better in case of the fusion. Consequently, the IBM could be designated as a multifunctional domain that was responsible for the activity enhancement, the stabilization, and the substrate binding of the rINUIBM. All these features obtained in this study make the rINUIBM become an attractive candidate for an efficient inulin hydrolysis.

Covalent immobilization of lysozyme on ethylene vinyl alcohol films for nonmigrating antimicrobial packaging applications.

Science.gov (United States)

Muriel-Galet, V; Talbert, J N; Hernandez-Munoz, P; Gavara, R; Goddard, J M

2013-07-10

The objective of this study was to develop a new antimicrobial film, in which lysozyme was covalently attached onto two different ethylene vinyl alcohol copolymers (EVOH 29 and EVOH 44). The EVOH surface was modified with UV irradiation treatment to generate carboxylic acid groups, and lysozyme was covalently attached to the functionalized polymer surface. Surface characterization of control and modified films was performed using attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) and dye assay. The value of protein loading after attachment on the surface was 8.49 μg protein/cm(2) and 5.74 μg protein/cm(2) for EVOH 29 and EVOH 44, respectively, after 10 min UV irradiation and bioconjugation. The efficacy of the EVOH-lysozyme films was assessed using Micrococcus lysodeikticus. The antimicrobial activity of the films was tested against Listeria monocytogenes and was similar to an equivalent amount of free enzyme. The reduction was 1.08 log for EVOH 29-lysozyme, 0.95 log for EVOH 44-lysozyme, and 1.34 log for free lysozyme. This work confirmed the successful use of lysozyme immobilization on the EVOH surface for antimicrobial packaging.

Penicillium subrubescens, a new species efficiently producing inulinase

NARCIS (Netherlands)

Mansouri, S.; Houbraken, J.; Samson, R.A.; Frisvad, J.C.; Christensen, M.; Tuthill, D.E.; Koutaniemi, S.; Hatakka, A.; Lankinen, P.

2013-01-01

Inulin is a reserve carbohydrate in about 15 % of the flowering plants and is accumulated in underground tubers of e.g. chicory, dahlia and Jerusalem artichoke. This carbohydrate consists of linear chains of β-(2,1)-linked fructose attached to a sucrose molecule. Inulinases hydrolyse inulin into

Covalent immobilization of lipase onto aminopropyl-functionalized hydroxyapatite-encapsulated-γ-Fe2O3 nanoparticles: A magnetic biocatalyst for interesterification of soybean oil.

Science.gov (United States)

Xie, Wenlei; Zang, Xuezhen

2017-07-15

Hydroxyapatite-encapsulated γ-Fe 2 O 3 nanoparticles were prepared, and lipase from Candida rugosa was then covalently bound onto the magnetic materials via covalent linkages. The magnetic carrier and immobilized lipase were characterized by enzyme activity assays, XRD, FT-IR, TEM, VSM and N 2 adsorption-desorption techniques. Results demonstrated that γ-Fe 2 O 3 nanoparticles were coated with the hydroxyapatite, and the lipase was indeed tethered to the magnetic carriers without damage to their structure. The immobilized lipase showed a strong magnetic responsiveness and displayed high catalytic activities towards the interesterification of soybean oil. The interesterified products were evaluated for their total fatty acid (FA) composition, slip melting point (SMP), iodine value, triacylglycerols (TAGs) profile and FA composition at sn-2 position in TAGs. The FA positional distributions and TAG species significantly changed after the enzymatic interesterification. Besides this, the interesterified products showed an obvious reduction in their SMP in comparison with the physical blends. Copyright © 2017 Elsevier Ltd. All rights reserved.

Enantioselective Synthesis of Various Cyanohydrins Using Covalently Immobilized Preparations of Hydroxynitrile Lyase from Prunus dulcis.

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Alagöz, Dilek; Tükel, S Seyhan; Yildirim, Deniz

2015-11-01

The carrier-based and carrier-free (cross-linked enzyme aggregate) covalent immobilizations of Prunus dulcis hydroxynitrile lyase were investigated. The immobilized preparations were tested for enantioselective carbon-carbon bond formation activity in the biphasic medium. Of the tested preparations, only cross-linked enzyme aggregate of P. dulcis hydroxynitrile lyase (PdHNL-CLEA) achieved the synthesis of (R)-mandelonitrile with 93% yield and 99% enantiopurity. PdHNL-CLEA was also used in the synthesis of various (R)-cyanohydrins from corresponding aldehydes/ketones and hydrocyanic acid. When 4-methoxybenzaldehyde, 4-methyl benzaldehyde, and 4-hydroxybenzaldehyde were used as substrates, the yield-enantiomeric excess of corresponding (R)-cyanohydrins were obtained as 95-95, 85-79, and 2-25%, respectively, after 96 h at pH 4.0 and 5 °C. For acetophenone, 4-fluoroacetophenone, 4-chloroacetophenone, 4-bromoacetophenone, and 4-iodoacetophenone, the yield-enantiomeric excess of corresponding (R)-cyanohydrins were 1-99, 20-84, 11-95, 5-99, and 3-24%, respectively at the same conditions. The results demonstrate PdHNL-CLEA can be effectively used in the synthesis of (R)-mandelonitrile.

Bioconversion of Agave tequilana fructans by exo-inulinases from indigenous Aspergillus niger CH-A-2010 enhances ethanol production from raw Agave tequilana juice.

Science.gov (United States)

Huitrón, Carlos; Pérez, Rosalba; Gutiérrez, Luís; Lappe, Patricia; Petrosyan, Pavel; Villegas, Jesús; Aguilar, Cecilia; Rocha-Zavaleta, Leticia; Blancas, Abel

2013-01-01

Agave tequilana fructans are the source of fermentable sugars for the production of tequila. Fructans are processed by acid hydrolysis or by cooking in ovens at high temperature. Enzymatic hydrolysis is considered an alternative for the bioconversion of fructans. We previously described the isolation of Aspergillus niger CH-A-2010, an indigenous strain that produces extracellular inulinases. Here we evaluated the potential application of A. niger CH-A-2010 inulinases for the bioconversion of A. tequilana fructans, and its impact on the production of ethanol. Inulinases were analyzed by Western blotting and thin layer chromatography. Optimal pH and temperature conditions for inulinase activity were determined. The efficiency of A. niger CH-A-2010 inulinases was compared with commercial enzymes and with acid hydrolysis. The hydrolysates obtained were subsequently fermented by Saccharomyces cerevisiae to determine the efficiency of ethanol production. Results indicate that A. niger CH-A-2010 predominantly produces an exo-inulinase activity. Optimal inulinase activity occurred at pH 5.0 and 50 °C. Hydrolysis of raw agave juice by CH-A-2010 inulinases yielded 33.5 g/l reducing sugars, compared with 27.3 g/l by Fructozyme(®) (Novozymes Corp, Bagsværd, Denmark) and 29.4 g/l by acid hydrolysis. After fermentation of hydrolysates, we observed that the conversion efficiency of sugars into ethanol was 97.5 % of the theoretical ethanol yield for enzymatically degraded agave juice, compared to 83.8 % for acid-hydrolyzed juice. These observations indicate that fructans from raw Agave tequilana juice can be efficiently hydrolyzed by using A. niger CH-A-2010 inulinases, and that this procedure impacts positively on the production of ethanol.

Covalent Immobilization of Candida rugosa Lipase at Alkaline pH and Their Application in the Regioselective Deprotection of Per-O-acetylated Thymidine

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Cintia W. Rivero

2016-08-01

Full Text Available Lipase from Candida rugosa (CRL was stabilized at alkaline pH to overcome the inactivation problem and was immobilized for the first time by multipoint covalent attachment on different aldehyde-activated matrices. PEG was used as a stabilizing agent on the activity of CRL. At these conditions, CRL maintained 50% activity at pH 10 after 17 h incubation in the presence of 40% (w/v of PEG, whereas the enzyme without additive was instantaneously inactive after incubation at pH 10. Thus, this enzyme was covalently immobilized at alkaline pH on three aldehyde-activated supports: aldehyde-activated Sepharose, aldehyde-activated Lewatit105 and heterofunctional aldehyde-activated EDA-Sepharose in high overall yields. Heterogeneous stable CRL catalysts at high temperature and solvent were obtained. The aldehyde-activated Sepharose-CRL preparation maintained 70% activity at 50 °C or 30% (v/v acetonitrile after 22 h and exhibited high regioselectivity in the deprotection process of per-O-acetylated thymidine, producing the 3′-OH-5′-OAc-thymidine in 91% yield at pH 5.

Bioelectrocatalytic and biosensing properties of horseradish peroxidase covalently immobilized on (3-aminopropyl)trimethoxysilane-modified titanate nanotubes

International Nuclear Information System (INIS)

Sovic, David; Gajovic, Andreja; Ivekovic, Damir

2011-01-01

Titanate nanotubes (TiNT) surface modified with (3-aminopropyl)trimethoxysilane were employed as a support for covalent immobilization of horseradish peroxidase (HRP) by using 1,4-benzoquinone as a coupling agent. Composite film-electrodes consisting of HRP-modified TiNT embedded into the porous carbon powder/Nafion matrix were fabricated and their applicability in direct bioelectrocatalytic reduction of H 2 O 2 and H 2 O 2 biosensing were investigated. An efficient direct electron transfer between the immobilized HRP molecules and the electrode was observed in the presence of H 2 O 2 at potentials lower than 600 mV (vs. Hg/Hg 2 Cl 2 /3.5 M KCl). For the HRP-TiNT-modified electrodes polarized at 0 mV, a linear dependence of the bioelectrocatalytic current on the concentration of H 2 O 2 was observed up to the concentration of H 2 O 2 equal to 10 μM, with the sensitivity of (1.10 ± 0.01) AM -1 cm -2 and the detection limit of 35 nM.

Data in support of covalent attachment of tyrosinase onto cyanuric chloride crosslinked magnetic nanoparticles

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Kourosh Abdollahi

2016-12-01

Full Text Available Preparation and characterization of cross linked amine-functionalized magnetic nanoparticles as an appropriate support for covalent immobilization on tyrosinase was presented in the study "Covalent immobilization of tyrosinase onto cyanuric chloride crosslinked amine-functionalized superparamagnetic nanoparticles: synthesis and characterization of the recyclable nanobiocatalyst" (Abdollahi et al., 2016 [1]. Herein, complementary data regarding X-ray powder diffraction (XRD to characterize the synthesized magnetic nanoparticles, and transmission electron microscopy (TEM to determine the size and morphology of tyrosinase immobilized magnetic nanoparticles (tyrosinase-MNPs were reported. The purification results of the extracted tyrosinase from mushroom Agaricus bisporus were provided in a purification table. The covalent immobilization of tyrosinase onto cyanuric chloride functionalized magnetic nanoparticles was proved by performing thermo-gravimetric and energy-dispersive X-ray spectroscopy analyses. The operational stability of immobilized tyrosinase was investigated by incubating tyrosinase-MNPs at different pH and temperatures.

Expression of exo-inulinase gene from Aspergillus niger 12 in E. coli strain Rosetta-gami B (DE3) and its characterization.

Science.gov (United States)

Yedahalli, Shreyas S; Rehmann, Lars; Bassi, Amarjeet

2016-05-01

Inulin is a linear carbohydrate polymer of fructose subunits (2-60) with terminal glucose units, produced as carbon storage in selected plants. It cannot directly be taken up by most microorganisms due to its large size, unless prior hydrolysis through inulinase enzymes occurs. The hydrolyzed inulin can be taken up by microbes and/or recovered and used industrially for the production of high fructose syrup, inulo-oligosaccharides, biofuel, and nutraceuticals. Cell-free enzymatic hydrolysis would be desirable for industrial applications, hence the recombinant expression, purification and characterization of an Aspergillus niger derived exo-inulinase was investigated in this study. The eukaroyototic exo-inulinase of Aspergillus niger 12 has been expressed, for the first time, in an E. coli strain [Rosetta-gami B (DE3)]. The molecular weight of recombinant exo-inulinase was estimated to be ∼81 kDa. The values of Km and Vmax of the recombinant exo-inulinase toward inulin were 5.3 ± 1.1 mM and 402.1 ± 53.1 µmol min(-1)  mg(-1) protein, respectively. Towards sucrose the corresponding values were 12.20 ± 1.6 mM and 902.8 ± 40.2 µmol min(-1)  mg(-1) protein towards sucrose. The S/I ratio was 2.24 ± 0.7, which is in the range of native inulinase. The optimum temperature and pH of the recombinant exo-inulinase towards inulin was 55°C and 5.0, while they were 50°C and 5.5 towards sucrose. The recombinant exo-inulinase activity towards inulin was enhanced by Cu(2+) and reduced by Fe(2+) , while its activity towards sucrose was enhanced by Co(2+) and reduced by Zn(2+) . © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:629-637, 2016. © 2016 American Institute of Chemical Engineers.

Diversity of inulinase-producing fungi associated with two Asteraceous plants, Pulicaria crispa (Forssk.) and Pluchea dioscoridis (L.) growing in an extreme arid environment

OpenAIRE

Khalil, Doaa M. A.; Massoud, Mohamed S.; Abdelrahman, Mostafa; El-Zayat, Soad A.; El-Sayed, Magdi A.

2018-01-01

Inulinases are potentially valuable enzymes catalyze the hydrolysis of plant’s inulin into high fructose syrups as sweetening ingredients for food industry and ethanol production. The high demands for inulinase enzymes have promoted interest in microbial inulinases as the most suitable approach for biosynthesis of fructose syrups from inulin. Arid land ecosystem represents a valuable bioresource for soil microbial diversity with unique biochemical and physiological properties. In the present ...

Biological methanol production by immobilized Methylocella tundrae using simulated biohythane as a feed.

Science.gov (United States)

Patel, Sanjay K S; Singh, Raushan K; Kumar, Ashok; Jeong, Jae-Hoon; Jeong, Seong Hun; Kalia, Vipin C; Kim, In-Won; Lee, Jung-Kul

2017-10-01

Biohythane may be used as an alternative feed for methanol production instead of costly pure methane. In this study, methanol production potential of Methylocella tundrae immobilized through covalent immobilization, adsorption, and encapsulation was evaluated. Cells covalently immobilized on groundnut shells and chitosan showed a relative methanol production potential of 83.9 and 91.6%, respectively, compared to that of free cells. The maximum methanol production by free cells and cells covalently immobilized on groundnut shells and chitosan was 6.73, 6.20, and 7.23mM, respectively, using simulated biohythane as a feed. Under repeated batch conditions of eight cycles, cells covalently immobilized on chitosan and groundnut shells, and cells encapsulated in sodium-alginate resulted in significantly higher cumulative methanol production of 37.76, 31.80, and 25.58mM, respectively, than free cells (18.57mM). This is the first report on immobilization of methanotrophs on groundnut shells and its application in methanol production using biohythane as a feed. Copyright © 2017 Elsevier Ltd. All rights reserved.

Computer-aided design of bromelain and papain covalent immobilization

OpenAIRE

Cutiño-Avila, Bessy; Gil Pradas, Dayrom; Aragón Abreu, Carlos; Fernández Marrero, Yuniel; Hernández de la Torre, Martha; Salas Sarduy, Emir; Chávez Planes, María de los Ángeles; Guisán Seijas, José Manuel; Díaz Brito, Joaquín; del Monte-Martínez, Alberto

2014-01-01

Enzymes as immobilized derivatives have been widely used in Food, Agrochemical, Pharmaceutical and Biotechnological industries. Protein immobilization is probably the most used technology to improve the operational stability of these molecules. Bromelain (Ananas comosus) and papain (Carica papaya) are cystein proteases extensively used as immobilized biocatalyst with several applications in therapeutics, racemic mixtures resolution, affinity chromatography and others industrial scenarios. The...

Enhanced Inulin Saccharification by Self-Produced Inulinase from a Newly Isolated Penicillium sp. and its Application in D-Lactic Acid Production.

Science.gov (United States)

Zheng, Zhaojuan; Xu, Qianqian; Liu, Peng; Zhou, Fan; Ouyang, Jia

2018-03-10

In order to find an alternative for commercial inulinase, a strain XL01 identified as Penicillium sp. was screened for inulinase production. The broth after cultivated was centrifuged, filtered, and used as crude enzyme for the following saccharification. At pH 5.0 and 50 °C, the crude enzyme released 84.9 g/L fructose and 20.7 g/L glucose from 120 g/L inulin in 72 h. In addition, simultaneous saccharification and fermentation of chicory flour for D-lactic acid production was carried out using the self-produced crude inulinase and Lactobacillus bulgaricus CGMCC 1.6970. A high D-lactic acid titer and productivity of 122.0 g/L and 1.69 g/(L h) was achieved from 120 g/L chicory flour in 72 h. The simplicity for inulinase production and the high efficiency for D-lactic acid fermentation provide a perspective and profitable industrial biotechnology for utilization of the inulin-rich biomass.

Covalent Immobilization of Enoxacin onto Titanium Implant Surfaces for Inhibiting Multiple Bacterial Species Infection and In Vivo Methicillin-Resistant Staphylococcus aureus Infection Prophylaxis.

Science.gov (United States)

Nie, Bin'en; Long, Teng; Ao, Haiyong; Zhou, Jianliang; Tang, Tingting; Yue, Bing

2017-01-01

Infection is one of the most important causes of titanium implant failure in vivo A developing prophylactic method involves the immobilization of antibiotics, especially vancomycin, onto the surface of the titanium implant. However, these methods have a limited effect in curbing multiple bacterial infections due to antibiotic specificity. In the current study, enoxacin was covalently bound to an amine-functionalized Ti surface by use of a polyethylene glycol (PEG) spacer, and the bactericidal effectiveness was investigated in vitro and in vivo The titanium surface was amine functionalized with 3-aminopropyltriethoxysilane (APTES), through which PEG spacer molecules were covalently immobilized onto the titanium, and then the enoxacin was covalently bound to the PEG, which was confirmed by X-ray photoelectron spectrometry (XPS). A spread plate assay, confocal laser scanning microscopy (CLSM), and scanning electron microscopy (SEM) were used to characterize the antimicrobial activity. For the in vivo study, Ti implants were inoculated with methicillin-resistant Staphylococcus aureus (MRSA) and implanted into the femoral medullary cavity of rats. The degree of infection was assessed by radiography, micro-computed tomography, and determination of the counts of adherent bacteria 3 weeks after surgery. Our data demonstrate that the enoxacin-modified PEGylated Ti surface effectively prevented bacterial colonization without compromising cell viability, adhesion, or proliferation in vitro Furthermore, it prevented MRSA infection of the Ti implants in vivo Taken together, our results demonstrate that the use of enoxacin-modified Ti is a potential approach to the alleviation of infections of Ti implants by multiple bacterial species. Copyright © 2016 American Society for Microbiology.

(Au/PANA/PVAc) nanofibers as a novel composite matrix for albumin and streptavidin immobilization

Energy Technology Data Exchange (ETDEWEB)

Golshaei, Rana [University of Kashan, Institute of Nano Science and Nano Technology, Kashan, P.O. Box 87317-51167, Islamic Republic of Iran (Iran, Islamic Republic of); Guler, Zeliha [Istanbul Technical University, Nanoscience and Nanoengineering, Maslak, Istanbul 34469 (Turkey); Sarac, Sezai A., E-mail: sarac@itu.edu.tr [Istanbul Technical University, Nanoscience and Nanoengineering, Maslak, Istanbul 34469 (Turkey); Istanbul Technical University, Department of Chemistry and Polymer Science and Technology, Maslak, Istanbul 34469 (Turkey)

2016-03-01

A novel electrospun nanofiber mat (Au/PANA/PVAc) consists of (Gold/Poly Anthranilic acid) (Au/PANA) core/shell nanostructures as a support material for protein immobilization that was developed and characterized by electrochemical impedance spectroscopy. In the core/shells, PANA served carboxyl groups (− COOH) for covalent protein immobilization and Au enhanced the electrochemical properties by acting as tiny conduction centers to facilitate electron transfer. Covalent immobilization of albumin and streptavidin as model proteins onto the (Au/PANA/PVAc) nanofibers was carried out by using 1-ethyl-3-(dimethyl-aminopropyl) carbodiimide hydrochloride (EDC)/N-hydroxyl succinimide (NHS) activation. PVAc nanofibers were compared with Au/PANA/PVAc nanofibers before and after protein immobilization. The successful covalent binding of both albumin and streptavidin onto (Au/PANA/PVAc) nanofibers was confirmed by FTIR-ATR, Electron Microscopy/Energy-Dispersive X-ray Spectroscopy SEM/EDX and Electrochemical impedance spectroscopy (EIS). The nanofibers became resistive due to protein immobilization and the higher charge transfer resistance was observed after higher amount of protein was immobilized. - Highlights: • Au/PANA/PVAc nanofibers with (COOH) groups as a suitable supports for covalent immobilization of proteins. • Increasing of the resistivity of the nanofibers after immobilization of the proteins. • Activation of Au/PANA/PVAc nanofibers by using EDC/NHS.

Characterization of two thermostable inulinases from Rhizopus oligosporus NRRL 2710

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Saleh A. Mohamed

2015-06-01

Full Text Available Two inulinases (Inu2 and Inu3 were purified from Rhizopus oligosporus NRRL 2710 by chromatography on DEAE-Sepharose and Sephacryl S-200 columns. The molecular weight of Inu2 and Inu3 were determined to be 76 and 30 kDa, respectively. Inu2 and Inu3 had the same pH optimum at 5.0, temperature optimum at 50 and 60 °C, and thermal stability up to 60 and 70 °C for 1 h, respectively. Inu2 and Inu3 had low km values (0.93 and 0.70 mM, respectively indicating the high affinity toward inulin. Mg2+, Ca2+, Zn2+ and EDTA did not significantly influence the enzyme activity. Ni2+, Cu2+, Fe2+ and Co2+ showed a partial inhibitory effect, and Hg2+ had a strong inhibitory effect. p-Chloromercuribenzoate had a partial inhibitory effect on Inu2. From these findings, R. oligosporus inulinases can be beneficial enzymes for industrial enzymatic production of high fructose syrup.

PVA-Glutaraldehyde as support for lectin immobilization and affinity chromatography

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Moacyr Jesus Barreto de Melo Rêgo

2016-12-01

Full Text Available Immobilized lectins are a powerful biotechnological tool for separation and isolation of glycoconjugates. In the present study, polyvinyl alcohol (PVA and glutaraldehyde (GA were used as a support for Concanavalin A (Con A covalent immobilization and for entrapment of Parkia pendula seed gum (PpeG. Con A immobilization yielded approximately 30% and 0.6 M glucose solution was the minimum concentration able to elute fetuin from column. PVA-GA-PpeG column was efficiently recognized by pure P. pendula lectin (PpeL. These findings indicate that PVA-GA interpenetrated network showed to be an efficient support for lectin covalent immobilization and as affinity chromatography matrix after trapping of PpeG.

An electrochemical sensor for warfarin determination based on covalent immobilization of quantum dots onto carboxylated multiwalled carbon nanotubes and chitosan composite film modified electrode

International Nuclear Information System (INIS)

Gholivand, Mohammad Bagher; Mohammadi-Behzad, Leila

2015-01-01

A method is described for the construction of a novel electrochemical warfarin sensor based on covalent immobilization of CdS-quantum dots (CdS-QDs) onto carboxylated multiwalled carbon nanotubes/chitosan (CS) composite film on the surface of a glassy carbon electrode. The CdS-QDs/CS/MWCNTs were characterized by field-emission scanning electron microscopy (FE-SEM), transmission electron microscopy (TEM), Fourier transform infra-red (FTIR) spectroscopy, XRD analysis and electrochemical impedance spectroscopy (EIS). The sensor showed optimum anodic stripping response within 90 s at an accumulation potential of 0.75 V. The modified electrode was used to detect the concentration of warfarin with a wide linear range of 0.05–80 μM and a detection limit (S/N = 3) of 8.5 nM. The proposed sensor has good storage stability, repeatability and reproducibility and was successfully applied for the determination of warfarin in real samples such as urine, serum and milk. - Highlights: • A new sensitive sensor for warfarin determination was developed. • The sensor was constructed based on covalent immobilization of CdS-QDs on the chitosan/MWCNTs/GCE. • The parameters affecting the stripping analysis of warfarin were optimized. • The proposed sensor is used for trace determination of warfarin in urine, serum and milk

An electrochemical sensor for warfarin determination based on covalent immobilization of quantum dots onto carboxylated multiwalled carbon nanotubes and chitosan composite film modified electrode

Energy Technology Data Exchange (ETDEWEB)

Gholivand, Mohammad Bagher, E-mail: mbgholivand2013@gmail.com; Mohammadi-Behzad, Leila

2015-12-01

A method is described for the construction of a novel electrochemical warfarin sensor based on covalent immobilization of CdS-quantum dots (CdS-QDs) onto carboxylated multiwalled carbon nanotubes/chitosan (CS) composite film on the surface of a glassy carbon electrode. The CdS-QDs/CS/MWCNTs were characterized by field-emission scanning electron microscopy (FE-SEM), transmission electron microscopy (TEM), Fourier transform infra-red (FTIR) spectroscopy, XRD analysis and electrochemical impedance spectroscopy (EIS). The sensor showed optimum anodic stripping response within 90 s at an accumulation potential of 0.75 V. The modified electrode was used to detect the concentration of warfarin with a wide linear range of 0.05–80 μM and a detection limit (S/N = 3) of 8.5 nM. The proposed sensor has good storage stability, repeatability and reproducibility and was successfully applied for the determination of warfarin in real samples such as urine, serum and milk. - Highlights: • A new sensitive sensor for warfarin determination was developed. • The sensor was constructed based on covalent immobilization of CdS-QDs on the chitosan/MWCNTs/GCE. • The parameters affecting the stripping analysis of warfarin were optimized. • The proposed sensor is used for trace determination of warfarin in urine, serum and milk.

Synthesis of Fructooligosaccharides from Sucrose Using Inulinase from Kluyveromyces marxianus

Directory of Open Access Journals (Sweden)

Francisco Maugeri

2007-01-01

Full Text Available Fructooligosaccharides (FOS from sucrose, new alternative sweeteners with functional properties, also called soluble fibers, have a number of desirable characteristics such as low calories, no cariogenicity, and safety for diabetics and Bifidus stimulating factor. Fructooligosaccharides are also known as prebiotics, since they stimulate probiotic organisms. The production, as well as the application of food-grade fructooligosaccharides, has increased rapidly during last years. In this work, experimental factorial design has been applied to optimize the fructooligosaccharide synthesis conditions by inulinase from Kluyveromyces marxianus var. bulgaricus. The studied variables were: temperature, pH, sucrose and enzyme concentrations. According to the results, only temperature and sucrose concentrations have shown to be significant parameters. The syntheses of the fructooligosaccharides were carried out on stirred reactor and packed bed reactors, using free and immobilized enzymes, with the best conditions obtained from the experimental design. It has been shown that there is no significant difference between these processes. The final sugar concentrations can be tailor made by varying residence time in the reactor to cope with the different standard needs in food industries. A typical solution product consists of a mixture of fructose (155 g/L, glucose (155 g/L, sucrose (132 g/L and fructooligosaccharides (50 g/L. These concentrations are suitable for applications in most food industries, in products such as sweets, candies, chocolates and yogurts. Besides, the prebiotic function of fructooligosaccharides as stimulants of the beneficial intestinal flora will give the product a functional and differentiated feature.

PRODUCTION, PROPERTIES AND APPLICATION OF SACCHAROMYCES CEREVISIAE VGSH-2 INULINASE

Directory of Open Access Journals (Sweden)

G. P. Shuvaeva

2014-01-01

Full Text Available Summary. Experimental data on an acid and thermal inactivation of a high refined inulinase (2,1-β-D- fructanfructanohydrolase, KF 3.2.17, produced by the race of Saccharomyces cerevisiae VGSh-2 yeast are presented. The strain of S. cerevisiae VGSh-2 was produced by the method of the induced mutagenesis and deposited to the collection of pure cultures of the chair of biochemistry and biotechnology of Voronezh state university of engineering technologies. The cells of source culture (S. cerevisiae XII were affected step-by-step by the ultra-violet radiation (UFR and UFR in a complex with a chemical mutagen (etilenimine. The culture was grown up by the method of liquid-phase deep cultivation on a constant nutrient medium. Refining conditions for inulinase are sorted out. Activity of enzyme dependence on physical and chemical factors (рН and temperature is obtained and numerical values of the main kinetic constants – Km and Vmax are determined. The structure of enzyme molecule is studied by an infrared-spectroscopy method: the type and relative quantity of elements of secondary structure of protein are defined. Substrate binding groups of the active center of an inulinase are found. The comparative analysis of the ability to hydrolysis of inulin in several enzyme preparations from Jerusalem artichoke and to the subsequent their fermentation by the VGSh-2 and XI S. cerevisiae yeasts is carried out. Optimum conditions of enzyme hydrolysis of inulin are selected. Research of the fermentation process of starchcontaining raw materials by yeasts of VGSh-2 and XI races is done. It is established that the using of VGSh-2 S. cerevisiae yeast for a grain wort and the Jerusalem artichoke fermentation, allows to increase an extraction of ethyl alcohol comparing to control race, to improve its quality characteristics, and also allows to predict the using of new race in the food industry for production ethanol from grain raw materials and a fermentation of

Covalent microcontact printing of proteins fro cell patterning

NARCIS (Netherlands)

Rozkiewicz, D.I.; Kraan, Yvonne M.; Werten, Marc W.T.; de Wolf, Frits A.; Subramaniam, Vinod; Ravoo, B.J.; Reinhoudt, David

2006-01-01

We describe a straightforward approach to the covalent immobilization of cytophilic proteins by microcontact printing, which can be used to pattern cells on substrates. Cytophilic proteins are printed in micropatterns on reactive self-assembled monolayers by using imine chemistry. An

Functionalization of multiwalled carbon nanotubes by microwave irradiation for lysozyme attachment: comparison of covalent and adsorption methods by kinetics of thermal inactivation

Science.gov (United States)

Puentes-Camacho, Daniel; Velázquez, Enrique F.; Rodríguez-Félix, Dora E.; Castillo-Ortega, Mónica; Sotelo-Mundo, Rogerio R.; del Castillo-Castro, Teresa

2017-12-01

Proteins suffer changes in their tertiary structure when they are immobilized, and enzymatic activity is affected due to the low biocompatibility of some supporting materials. In this work immobilization of lysozyme on carbon nanotubes previously functionalized by microwave irradiation was studied. The effectiveness of the microwave-assisted acid treatment of carbon nanotubes was evaluated by XPS, TEM, Raman and FTIR spectroscopy. The carboxylic modification of nanotube surfaces by this fast, simple and feasible method allowed the physical adsorption and covalent linking of active lysozyme onto the carbonaceous material. Thermal inactivation kinetics, thermodynamic parameters and storage stability were studied for adsorbed and covalent enzyme complexes. A major stability was found for lysozyme immobilized by the covalent method, the activation energy for inactivation of the enzyme was higher for the covalent method and it was stable after 50 d of storage at 4 °C. The current study highlights the effect of protein immobilization method on the biotechnological potential of nanostructured biocatalysts.

Computer-aided design of bromelain and papain covalent immobilization

OpenAIRE

Bessy Cutiño-Avila; Dayrom Gil Pradas; Carlos Aragón Abreu; Yuniel Fernández Marrero; Martha Hernández de la Torre; Emir Salas Sarduy; María de los Ángeles Chávez Planes; José Manuel Guisán Seijas; Joaquín Díaz Brito; Alberto del Monte-Martínez

2014-01-01

Título en español: Diseño asistido por computadora de la inmovilización covalente de bromelina y papaína. Título corto: Computer-aided design of bromelain and papain.  Abstract: Enzymes as immobilized derivatives have been widely used in Food, Agrochemical, Pharmaceutical and Biotechnological industries. Protein immobilization is probably the most used technology to improve the operational stability of these molecules. Bromelain (Ananas comosus) and papain (Carica papaya) are cystein pr...

Evaluation of the number of ionogenic groups of inulinase by acid-base titration.

Science.gov (United States)

Kovaleva, T A; Holyavka, M G; Rezvan, S G; Kozhedub, S V

2008-06-01

Acid base titration showed that Aspergillus awamori inulinase includes 178 asparaginic and glutamic acid residues, 20 histidine, 10 serine, and 34 lysine and tyrosine residues. Denaturation temperature for this enzyme was calculated using analysis of the proportion of stabilizing and destabilizing amino acids in the molecule.

Radical covalent organic frameworks: a general strategy to immobilize open-accessible polyradicals for high-performance capacitive energy storage.

Science.gov (United States)

Xu, Fei; Xu, Hong; Chen, Xiong; Wu, Dingcai; Wu, Yang; Liu, Hao; Gu, Cheng; Fu, Ruowen; Jiang, Donglin

2015-06-01

Ordered π-columns and open nanochannels found in covalent organic frameworks (COFs) could render them able to store electric energy. However, the synthetic difficulty in achieving redox-active skeletons has thus far restricted their potential for energy storage. A general strategy is presented for converting a conventional COF into an outstanding platform for energy storage through post-synthetic functionalization with organic radicals. The radical frameworks with openly accessible polyradicals immobilized on the pore walls undergo rapid and reversible redox reactions, leading to capacitive energy storage with high capacitance, high-rate kinetics, and robust cycle stability. The results suggest that channel-wall functional engineering with redox-active species will be a facile and versatile strategy to explore COFs for energy storage. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Covalent immobilization of cholesterol esterase and cholesterol oxidase on polyaniline films for application to cholesterol biosensor

Energy Technology Data Exchange (ETDEWEB)

Singh, Suman [Biomolecular Electronics and Conducting Polymer Research Group, National Physical Laboratory, Dr. K. S. Krishnan Marg, New Delhi-110012 (India); Solanki, Pratima R. [Biomolecular Electronics and Conducting Polymer Research Group, National Physical Laboratory, Dr. K. S. Krishnan Marg, New Delhi-110012 (India); Pandey, M.K. [Biomolecular Electronics and Conducting Polymer Research Group, National Physical Laboratory, Dr. K. S. Krishnan Marg, New Delhi-110012 (India); Malhotra, B.D. [Biomolecular Electronics and Conducting Polymer Research Group, National Physical Laboratory, Dr. K. S. Krishnan Marg, New Delhi-110012 (India)]. E-mail: bansi@mail.nplindia.ernet.in

2006-05-24

Cholesterol esterase (ChEt) and cholesterol oxidase (ChOx) have been covalently immobilized on electrochemically prepared polyaniline (PANI) films. These PANI/ChEt/ChOx enzyme films have been characterized using UV-visible, Fourier transform infrared (FTIR) spectroscopy and scanning electron microscopy (SEM). Electrochemical behavior of these films has been studied using cyclic voltammetry (CV) and amperometric techniques, respectively. The PANI/ChEt/ChOx enzyme films show broad oxidation peak from 0.2 to 0.5 V. These PANI/ChEt/ChOx biosensing electrodes have a response time of about 40 s, linearity from 50 to 500 mg/dl of cholesterol oleate concentration. These PANI/ChEt/ChOx films are thermally stable up to 46 deg. C. This polyaniline based cholesterol biosensor has optimum pH in the range of 6.5-7.5, sensitivity as 7.5 x 10{sup -4} nA/mg dl and a lifetime of about 6 weeks.

Covalent immobilization of invertase on PAMAM-dendrimer modified superparamagnetic iron oxide nanoparticles

International Nuclear Information System (INIS)

Uzun, K.; Cevik, E.; Senel, M.; Soezeri, H.; Baykal, A.; Abasiyanik, M. F.; Toprak, M. S.

2010-01-01

In this study, polyamidoamine (PAMAM) dendrimer was synthesized on the surface of superparamagnetite nanoparticles to enhance invertase immobilization. The amount of immobilized enzyme on the surface-hyperbranched magnetite nanoparticle was up to 2.5 times (i.e., 250%) as much as that of magnetite nanoparticle modified with only amino silane. Maximum reaction rate (V max ) and Michaelis-Menten constant (K m ) were determined for the free and immobilized enzymes. Various characteristics of immobilized invertase such as; the temperature activity, thermal stability, operational stability, and storage stability were evaluated and results revealed that stability of the enzyme is improved upon immobilization.

Optimizing culture conditions for production of intra and extracellular inulinase and invertase from Aspergillus niger ATCC 20611 by response surface methodology (RSM).

Science.gov (United States)

Dinarvand, Mojdeh; Rezaee, Malahat; Foroughi, Majid

The aim of this study was obtain a model that maximizes growth and production of inulinase and invertase by Aspergillus niger ATCC 20611, employing response surface methodology (RSM). The RSM with a five-variable and three-level central composite design (CCD) was employed to optimize the medium composition. Results showed that the experimental data could be appropriately fitted into a second-order polynomial model with a coefficient of determination (R 2 ) more than 0.90 for all responses. This model adequately explained the data variation and represented the actual relationships between the parameters and responses. The pH and temperature value of the cultivation medium were the most significant variables and the effects of inoculum size and agitation speed were slightly lower. The intra-extracellular inulinase, invertase production and biomass content increased 10-32 fold in the optimized medium condition (pH 6.5, temperature 30°C, 6% (v/v), inoculum size and 150rpm agitation speed) by RSM compared with medium optimized through the one-factor-at-a-time method. The process development and intensification for simultaneous production of intra-extracellular inulinase (exo and endo inulinase) and invertase from A. niger could be used for industrial applications. Copyright © 2017 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

Immobilization of Chloroperoxidase on Aminopropyl-Glass

Science.gov (United States)

Kadima, Tenshuk A.; Pickard, Michael A.

1990-01-01

Chloroperoxidase (CPO) purified from Caldariomyces fumago CMI 89362 was covalently bound to aminopropyl-glass by using a modification of an established method. Acid-washed glass was derivatized by using aminopropyltriethoxysilane, and the enzyme was ionically bound at low ionic strength. Further treatment with glutaraldehyde covalently linked the enzyme to the glass beads in an active form. No elution of bound activity from glass beads could be detected with a variety of washings. The loading of enzyme protein to the glass beads was highest, 100 mg of CPO per g of glass, at high reaction ratios of CPO to glass, but the specific activity of the immobilized enzyme was highest, 36% of theoretical, at low enzyme-to-carrier ratios. No differences in the properties of the soluble and immobilized enzymes could be detected by a number of criteria: their pH-activity and pH-stability profiles were similar, as were their thermal stabilities. After five uses, the immobilized enzyme retained full activity between pH 6.0 and 6.7. PMID:16348352

Lipase immobilization and production of fatty acid methyl esters from canola oil using immobilized lipase

International Nuclear Information System (INIS)

Yuecel, Yasin; Demir, Cevdet; Dizge, Nadir; Keskinler, Buelent

2011-01-01

Lipase enzyme from Aspergillus oryzae (EC 3.1.1.3) was immobilized onto a micro porous polymeric matrix which contains aldehyde functional groups and methyl esters of long chain fatty acids (biodiesel) were synthesized by transesterification of crude canola oil using immobilized lipase. Micro porous polymeric matrix was synthesized from styrene-divinylbenzene (STY-DVB) copolymers by using high internal phase emulsion technique and two different lipases, Lipozyme TL-100L ® and Novozym 388 ® , were used for immobilization by both physical adsorption and covalent attachment. Biodiesel production was carried out with semi-continuous operation. Methanol was added into the reactor by three successive additions of 1:4 M equivalent of methanol to avoid enzyme inhibition. The transesterification reaction conditions were as follows: oil/alcohol molar ratio 1:4; temperature 40 o C and total reaction time 6 h. Lipozyme TL-100L ® lipase provided the highest yield of fatty acid methyl esters as 92%. Operational stability was determined with immobilized lipase and it indicated that a small enzyme deactivation occurred after used repeatedly for 10 consecutive batches with each of 24 h. Since the process is yet effective and enzyme does not leak out from the polymer, the method can be proposed for industrial applications. -- Research highlights: → Lipozyme TL-100L and Novozym 388 were immobilized onto micro porous polymeric matrix by both physical adsorption and covalent linking. → Immobilized enzymes were used for synthesis of fatty acid methyl esters by transesterification of canola oil and methanol using semi-continuous operation system. → According to chromatographic analysis, Lipase Lipozyme TL-100L resulted in the highest yield of methyl ester as 92%.

Polyethyleneimine-modified superparamagnetic Fe3O4 nanoparticles for lipase immobilization: Characterization and application

International Nuclear Information System (INIS)

Khoobi, Mehdi; Motevalizadeh, Seyed Farshad; Asadgol, Zahra; Forootanfar, Hamid; Shafiee, Abbas; Faramarzi, Mohammad Ali

2015-01-01

Magnetically separable nanospheres consisting of polyethyleneimine (PEI) and succinated PEI grafted on silica coated magnetite (Fe 3 O 4 ) were prepared and characterized using Fourier transform infrared spectroscopy, thermogravimetric analysis, X-ray diffraction, vibrating sample magnetometer, scanning electron microscopy and transmission electron microscopy. The prepared magnetic nanoparticles were then applied for physical adsorption or covalent attachment of Thermomyces lanuginosa lipase (TLL) via glutaraldehyde or hexamethylene diisocyanate. The reusability, storage, pH and thermal stabilities of the immobilized enzymes compared to that of free lipase were examined. The obtained results showed that the immobilized lipase on MNPs@PEI-GLU was the best biocatalyst which retained 80% of its initial activity after 12 cycles of application. The immobilized lipase on the selected support (MNPs@PEI-GLU) was also applied for the synthesis of ethyl valerate. Following 24 h incubation of the immobilized lipase on the selected support in n-hexane and solvent free media, the esterification percentages were 72.9% and 28.9%, respectively. - Graphical abstract: A schematic of the preparation of PEI- and succinated PEI-grafted Fe 3 O 4 MNPs (MNPs@PEI) and the immobilization of lipase by covalent bonding and adsorption. - Highlights: • Functionalized polyethylenimine-grafted magnetic nanoparticles were synthesized. • The prepared supports were fully characterized by various analysis methods. • Lipase was immobilized on the nanostructures by adsorption and covalent attachment. • Immobilized lipase produced ethyl valerate in solvent free medium

[Native, modified, and immobilized chymotrypsin in chaotropic media. Stabilization limits].

Science.gov (United States)

Panova, A A; Levitskiĭ, V Iu; Mozhaev, V V

1994-07-01

To stabilize alpha-chymotrypsin against irreversible thermal inactivation at high temperatures, methods of covalent modification and multi-point immobilization in combination with the addition of salting-in compounds were used. The upper limit of the protein stability proved to be the same for a combination of the modification and salting-in media and for each of these methods separately. The limit of stabilization reached by means of covalent immobilization is higher than the limit of stabilization reached by two other methods. The greatest stabilization of immobilized alpha-chymotrypsin by the salting-in media (a 10000 fold increase in the native enzyme's stability level) takes place only in the case of the protein with the minimum number of bonds with the support. Stabilization of the enzyme by these methods is explained in terms of the suppression of the conformational inactivation processes.

Mechanisms for Covalent Immobilization of Horseradish Peroxidase on Ion-Beam-Treated Polyethylene

Directory of Open Access Journals (Sweden)

Alexey V. Kondyurin

2012-01-01

Full Text Available The surface of polyethylene was modified by plasma immersion ion implantation. Structure changes including carbonization and oxidation were observed. High surface energy of the modified polyethylene was attributed to the presence of free radicals on the surface. The surface energy decay with storage time after treatment was explained by a decay of the free radical concentration while the concentration of oxygen-containing groups increased with storage time. Horseradish peroxidase was covalently attached onto the modified surface by the reaction with free radicals. Appropriate blocking agents can block this reaction. All aminoacid residues can take part in the covalent attachment process, providing a universal mechanism of attachment for all proteins. The native conformation of attached protein is retained due to hydrophilic interactions in the interface region. The enzymatic activity of covalently attached protein remained high. The long-term activity of the modified layer to attach protein is explained by stabilisation of unpaired electrons in sp2 carbon structures. A high concentration of free radicals can give multiple covalent bonds to the protein molecule and destroy the native conformation and with it the catalytic activity. The universal mechanism of protein attachment to free radicals could be extended to various methods of radiation damage of polymers.

Stabilization of penicillin G acylase by immobilization on glutaraldehyde-activated chitosan

Directory of Open Access Journals (Sweden)

W. S. Adriano

2005-12-01

Full Text Available The objective of this work was to study enzyme immobilization on chitosan activated with glutaraldehyde, aiming to produce a cheap biocatalyst. Two different immobilization strategies were studied: one-point and multipoint covalent attachment to the solid matrix. The multipoint covalent attachment derivative had an 82% immobilization yield. It was 4.9-fold more stable than the free enzyme at 50°C and 4.5-fold more stable than soluble enzyme at pH 10.0. The one-point derivative had an 85% immobilization yield. It was 2.7-fold more stable than the free enzyme at 50°C and 3.8-fold more stable than soluble PGA at pH 10.0. Results indicated that chitosan can be loaded with PGA above 330 IU/g. Intraparticle diffusive effects, however, limited hydrolysis of penicillin G catalyzed by those derivatives at 37°C and 25°C. Operational stability assays were performed and the multipoint derivative exhibited a half-life of 40 hours.

Recent Developments in the Site-Specific Immobilization of Proteins onto Solid Supports

Energy Technology Data Exchange (ETDEWEB)

Camarero, J A

2007-02-21

Immobilization of proteins onto surfaces is of great importance in numerous applications, including protein analysis, drug screening, and medical diagnostics, among others. The success of all these technologies relies on the immobilization technique employed to attach a protein to the corresponding surface. Non-specific physical adsorption or chemical cross-linking with appropriate surfaces results in the immobilization of the protein in random orientations. Site-specific covalent attachment, on the other hand, leads to molecules being arranged in a definite, orderly fashion and allows the use of spacers and linkers to help minimize steric hindrances between the protein and the surface. The present work reviews the latest chemical and biochemical developments for the site-specific covalent attachment of proteins onto solid supports.

Immobilizing Biomolecules Near the Diffraction Limit

DEFF Research Database (Denmark)

Skovsen, Esben; Petersen, Maria Teresa Neves; Gennaro, Ane Kold Di

2009-01-01

Our group has previously shown that biomolecules containing disulfide bridges in close proximity to aromatic residues can be immobilized, through covalent bonds, onto thiol derivatized surfaces upon UV excitation of the aromatic residue(s). We have also previously shown that our new technology ca...

Immobilization of collagen peptide on dialdehyde bacterial cellulose nanofibers via covalent bonds for tissue engineering and regeneration

Directory of Open Access Journals (Sweden)

Wen XX

2015-07-01

Full Text Available Xiaoxiao Wen,1 Yudong Zheng,1 Jian Wu,2 Lu-Ning Wang,1 Zhenya Yuan,1 Jiang Peng,3 Haoye Meng3 1School of Materials Science and Engineering, University of Science and Technology Beijing, Beijing, People’s Republic of China; 2Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences, Soochow, People’s Republic of China; 3Institute of Orthopedics, Chinese PLA General Hospital, Beijing, People’s Republic of China Abstract: Bacterial cellulose (BC is an alternative nanostructured biomaterial to be utilized for a wide range of biomedical applications. Because of its low bioactivity, which restricted its practical application, collagen and collagen hydrolysate were usually composited into BC. It is necessary to develop a new method to generate covalent bonds between collagen and cellulose to improve the immobilization of collagen on BC. This study describes a facile dialdehyde BC/collagen peptide nanocomposite. BC was oxidized into dialdehyde bacterial cellulose (DBC by regioselective oxidation, and then composited with collagen peptide (Col-p via covalent bonds to form Schiff’s base type compounds, which was demonstrated by the results of microstructures, contact angle, Col-p content, and peptide-binding ratio. The peptide-binding ratio was further affected by the degree of oxidation, pH value, and zeta potential. In vitro desorption measurement of Col-p suggested a controlled release mechanism of the nanocomposite. Cell tests indicated that the prepared DBC/Col-p composite was bioactive and suitable for cell adhesion and attachment. This work demonstrates that the DBC/Col-p composite is a promising material for tissue engineering and regeneration. Keywords: bacterial cellulose, dialdehyde cellulose, collagen peptide, composite materials, cytoactivity 

Approaching Immobilization of Enzymes onto Open Porous Basotect®

Directory of Open Access Journals (Sweden)

Peter J. Allertz

2017-11-01

Full Text Available For the first time, commercial macroporous melamine formaldehyde foam Basotect® (BT was used as a basic carrier material for both adsorptive and covalent enzyme immobilization. In order to access inherent amino groups, the Basotect® surface was pretreated with hydrochloric acid. The resulting material revealed 6 nmol of superficial amino groups per milligram Basotect®. Different optimized strategies for tethering the laccase from Trametes versicolor and the lipase from Thermomyces lanuginosus onto the pre-treated Basotect® surface were studied. Particularly, for covalent immobilization, two different strategies were pursued: lipase was tethered via a cross-linking method using 1-ethyl-3-(3-dimethylaminopropylcarbodiimide, and laccase was bound after functionalizing Basotect® with hydrophilic copolymer poly(ethylene-alt-maleic anhydride (PEMA. Prior to laccase immobilization, the PEMA coating of Basotect® was verified by ATR-FTIR analysis. Subsequent quantification of available high-reactive PEMA anhydride moieties revealed an amount of 1028 ± 73 nmol per mg Basotect®. The surface-bound enzyme amounts were quantified as 4.1–5.8 μg per mg Basotect®. A theoretical surface-covered enzyme mass for the ideal case that an enzyme monolayer was immobilized onto the Basotect® surface was calculated and compared to the amount of adsorptive and covalently bound enzymes before and after treatment with SDS. Furthermore, the enzyme activities were determined for the different immobilization approaches, and the stability during storage over time and against sodium dodecyl sulfate treatment was monitored. Additionally, PEMA-BT-bound laccase was tested for the elimination of anthropogenic micropollutant bisphenol A from contaminated water in a cost-effective and environmentally-friendly way and resulted in a degradation rate higher than 80%.

Isolation of a Kluyveromyces fragilis derepressed mutant hyperproducer of inulinase for ethanol production from Jerusalem artichoke

Energy Technology Data Exchange (ETDEWEB)

Bourgi, J.; Guirand, J.P.; Galzy, P.

1986-01-01

A paritally derepressed mutant of Kluyveromyces fragilis showing hyperproduction of inulinase was isolated by means of ethylmethanesulfonate mutation followed by a 2-deoxyglucose selection. This mutant is suitable for the fermentation of inulin and Jerusalem artichoke extracts containing large amounts of inulin high polyfructosans type (early extracts).

Non-covalently functionalized carbon nanostructures for synthesizing carbon-based hybrid nanomaterials.

Science.gov (United States)

Li, Haiqing; Song, Sing I; Song, Ga Young; Kim, Il

2014-02-01

Carbon nanostructures (CNSs) such as carbon nanotubes, graphene sheets, and nanodiamonds provide an important type of substrate for constructing a variety of hybrid nanomaterials. However, their intrinsic chemistry-inert surfaces make it indispensable to pre-functionalize them prior to immobilizing additional components onto their surfaces. Currently developed strategies for functionalizing CNSs include covalent and non-covalent approaches. Conventional covalent treatments often damage the structure integrity of carbon surfaces and adversely affect their physical properties. In contrast, the non-covalent approach offers a non-destructive way to modify CNSs with desired functional surfaces, while reserving their intrinsic properties. Thus far, a number of surface modifiers including aromatic compounds, small-molecular surfactants, amphiphilic polymers, and biomacromolecules have been developed to non-covalently functionalize CNS surfaces. Mediated by these surface modifiers, various functional components such as organic species and inorganic nanoparticles were further decorated onto their surfaces, resulting in versatile carbon-based hybrid nanomaterials with broad applications in chemical engineering and biomedical areas. In this review, the recent advances in the generation of such hybrid nanostructures based on non-covalently functionalized CNSs will be reviewed.

Polyethyleneimine-modified superparamagnetic Fe{sub 3}O{sub 4} nanoparticles for lipase immobilization: Characterization and application

Energy Technology Data Exchange (ETDEWEB)

Khoobi, Mehdi; Motevalizadeh, Seyed Farshad [Department of Medicinal Chemistry, Faculty of Pharmacy and Pharmaceutical Sciences Research Center, Tehran University of Medical Sciences, Tehran 1417614411 (Iran, Islamic Republic of); Asadgol, Zahra [Department of Pharmaceutical Biotechnology, Faculty of Pharmacy and Biotechnology Research Center, Tehran University of Medical Sciences, P.O. Box 14155-6451, Tehran 1417614411 (Iran, Islamic Republic of); Forootanfar, Hamid [Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Kerman University of Medical Sciences, Kerman (Iran, Islamic Republic of); Shafiee, Abbas, E-mail: ashafiee@ams.ac.ir [Department of Medicinal Chemistry, Faculty of Pharmacy and Pharmaceutical Sciences Research Center, Tehran University of Medical Sciences, Tehran 1417614411 (Iran, Islamic Republic of); Faramarzi, Mohammad Ali, E-mail: faramarz@tums.ac.ir [Department of Pharmaceutical Biotechnology, Faculty of Pharmacy and Biotechnology Research Center, Tehran University of Medical Sciences, P.O. Box 14155-6451, Tehran 1417614411 (Iran, Islamic Republic of)

2015-01-15

Magnetically separable nanospheres consisting of polyethyleneimine (PEI) and succinated PEI grafted on silica coated magnetite (Fe{sub 3}O{sub 4}) were prepared and characterized using Fourier transform infrared spectroscopy, thermogravimetric analysis, X-ray diffraction, vibrating sample magnetometer, scanning electron microscopy and transmission electron microscopy. The prepared magnetic nanoparticles were then applied for physical adsorption or covalent attachment of Thermomyces lanuginosa lipase (TLL) via glutaraldehyde or hexamethylene diisocyanate. The reusability, storage, pH and thermal stabilities of the immobilized enzymes compared to that of free lipase were examined. The obtained results showed that the immobilized lipase on MNPs@PEI-GLU was the best biocatalyst which retained 80% of its initial activity after 12 cycles of application. The immobilized lipase on the selected support (MNPs@PEI-GLU) was also applied for the synthesis of ethyl valerate. Following 24 h incubation of the immobilized lipase on the selected support in n-hexane and solvent free media, the esterification percentages were 72.9% and 28.9%, respectively. - Graphical abstract: A schematic of the preparation of PEI- and succinated PEI-grafted Fe{sub 3}O{sub 4} MNPs (MNPs@PEI) and the immobilization of lipase by covalent bonding and adsorption. - Highlights: • Functionalized polyethylenimine-grafted magnetic nanoparticles were synthesized. • The prepared supports were fully characterized by various analysis methods. • Lipase was immobilized on the nanostructures by adsorption and covalent attachment. • Immobilized lipase produced ethyl valerate in solvent free medium.

Immobilization of biomolecules onto surfaces according to ultraviolet light diffraction patterns

DEFF Research Database (Denmark)

Petersen, Steffen B.; Gennaro, Ane Kold Di; Neves Petersen, Teresa

2010-01-01

We developed a method for immobilization of biomolecules onto thiol functionalized surfaces according to UV diffraction patterns. UV light-assisted molecular immobilization proceeds through the formation of free, reactive thiol groups that can bind covalently to thiol reactive surfaces. We demons......, with a fine structured interference pattern superimposed. (C) 2010 Optical Society of America...

In situ ellipsometric study of surface immobilization of flagellar filaments

Energy Technology Data Exchange (ETDEWEB)

Kurunczi, S., E-mail: kurunczi@mfa.kfki.hu [Department of Photonics, Research Institute for Technical Physics and Materials Science, H-1121, Konkoly Thege Miklos ut 29-33, Budapest (Hungary); Nemeth, A.; Huelber, T. [Department of Photonics, Research Institute for Technical Physics and Materials Science, H-1121, Konkoly Thege Miklos ut 29-33, Budapest (Hungary); Kozma, P. [Department of Photonics, Research Institute for Technical Physics and Materials Science, H-1121, Konkoly Thege Miklos ut 29-33, Budapest (Hungary); Department of Nanotechnology, Research Institute of Chemical and Process Engineering, Faculty of Information Technology, University of Pannonia, Egyetem u. 10, Veszprem, H-8200 (Hungary); Petrik, P. [Department of Photonics, Research Institute for Technical Physics and Materials Science, H-1121, Konkoly Thege Miklos ut 29-33, Budapest (Hungary); Jankovics, H. [Department of Nanotechnology, Research Institute of Chemical and Process Engineering, Faculty of Information Technology, University of Pannonia, Egyetem u. 10, Veszprem, H-8200 (Hungary); Sebestyen, A. [Department of Photonics, Research Institute for Technical Physics and Materials Science, H-1121, Konkoly Thege Miklos ut 29-33, Budapest (Hungary); Department of Nanotechnology, Research Institute of Chemical and Process Engineering, Faculty of Information Technology, University of Pannonia, Egyetem u. 10, Veszprem, H-8200 (Hungary); Vonderviszt, F. [Department of Photonics, Research Institute for Technical Physics and Materials Science, H-1121, Konkoly Thege Miklos ut 29-33, Budapest (Hungary); Department of Nanotechnology, Research Institute of Chemical and Process Engineering, Faculty of Information Technology, University of Pannonia, Egyetem u. 10, Veszprem, H-8200 (Hungary); Institute of Enzymology, Karolina ut 29-33, Budapest, H-1113 (Hungary); and others

2010-10-15

Protein filaments composed of thousands of subunits are promising candidates as sensing elements in biosensors. In this work in situ spectroscopic ellipsometry is applied to monitor the surface immobilization of flagellar filaments. This study is the first step towards the development of layers of filamentous receptors for sensor applications. Surface activation is performed using silanization and a subsequent glutaraldehyde crosslinking. Structure of the flagellar filament layers immobilized on activated and non-activated Si wafer substrates is determined using a two-layer effective medium model that accounted for the vertical density distribution of flagellar filaments with lengths of 300-1500 nm bound to the surface. The formation of the first interface layer can be explained by the multipoint covalent attachment of the filaments, while the second layer is mainly composed of tail pinned filaments floating upwards with the free parts. As confirmed by atomic force microscopy, covalent immobilization resulted in an increased surface density compared to absorption.

activity of enzyme trypsin immobilized onto macroporous poly(epoxy

African Journals Online (AJOL)

dell

consequential effects of covalent immobilization. EXPERIMENTAL. Materials .... immersed into water bath. ... storage stability of the enzyme was studied ... pore size range of about 10 to 150 µm. ... figures, the differences in activities (slopes.

Fermentative hydrogen production from Jerusalem artichoke by Clostridium tyrobutyricum expressing exo-inulinase gene.

Science.gov (United States)

Jiang, Ling; Wu, Qian; Xu, Qing; Zhu, Liying; Huang, He

2017-08-11

Clostridium tyrobutyricum ATCC25755 has been reported as being able to produce significant quantities of hydrogen. In this study, the exo-inulinase encoding gene cloned from Paenibacillus polymyxa SC-2 was into the expression plasmid pSY6 and expressed in the cells of C. tyrobutyricum. The engineered C. tyrobutyricum strain efficiently fermented the inulin-type carbohydrates from Jerusalem artichoke, without any pretreatment being necessary for the production of hydrogen. A comparatively high hydrogen yield (3.7 mol/mol inulin-type sugar) was achieved after 96 h in a batch process with simultaneous saccharification and fermentation (SSF), with an overall volumetric productivity rate of 620 ± 60 mL/h/L when the initial total sugar concentration of the inulin extract was increased to 100 g/L. Synthesis of inulinase in the batch SSF culture was closely associated with strain growth until the end of the exponential phase, reaching a maximum activity of 28.4 ± 0.26 U/mL. The overall results show that the highly productive and abundant biomass crop Jerusalem artichoke can be a good substrate for hydrogen production, and that the application of batch SSF for its conversion has the potential to become a cost-effective process in the near future.

Biosensor based on laccase immobilized on plasma polymerized allylamine/carbon electrode

Energy Technology Data Exchange (ETDEWEB)

Ardhaoui, Malika, E-mail: malika.ardhaoui@ucd.ie [Laboratoire de Génie des Procédés Plasma et Traitements de Surface, Université Pierre et Marie Curie-Chimie ParisTech, 11 rue Pierre et Marie Curie, 75231 Paris (France); Laboratoire Charles Friedel, CNRS UMR 7223, Chimie ParisTech, 11 rue Pierre et Marie Curie, 75231 Paris Cedex 05 (France); Surface Engineering Research Group, School of Electrical, Electronic and Mechanical Engineering, University College Dublin, Belfield, Dublin 4 (Ireland); Bhatt, Sudhir [Laboratoire de Génie des Procédés Plasma et Traitements de Surface, Université Pierre et Marie Curie-Chimie ParisTech, 11 rue Pierre et Marie Curie, 75231 Paris (France); Zheng, Meihui [Laboratoire Charles Friedel, CNRS UMR 7223, Chimie ParisTech, 11 rue Pierre et Marie Curie, 75231 Paris Cedex 05 (France); Dowling, Denis [Surface Engineering Research Group, School of Electrical, Electronic and Mechanical Engineering, University College Dublin, Belfield, Dublin 4 (Ireland); Jolivalt, Claude [Laboratoire Charles Friedel, CNRS UMR 7223, Chimie ParisTech, 11 rue Pierre et Marie Curie, 75231 Paris Cedex 05 (France); Khonsari, Farzaneh Arefi [Laboratoire de Génie des Procédés Plasma et Traitements de Surface, Université Pierre et Marie Curie-Chimie ParisTech, 11 rue Pierre et Marie Curie, 75231 Paris (France)

2013-08-01

In this work, a simple and rapid method was used to functionalize carbon electrode in order to efficiently immobilize laccase for biosensor application. A stable allylamine coating was deposited using a low pressure inductively excited RF tubular plasma reactor under mild plasma conditions (low plasma power (10 W), few minutes) to generate high density amine groups (N/C ratio up to 0.18) on rough carbon surface electrodes. The longer was the allylamine plasma deposition time; the better was the surface coverage. Laccase from Trametes versicolor was physisorbed and covalently bound to these allylamine modified carbon surfaces. The laccase activities and current outputs measured in the presence of 2,2′-azinobis-(3-ethylbenzothiazole-6-sulfonic acid) (ABTS) showed that the best efficiency was obtained for electrode plasma coated during 30 min. They showed also that for all the tested electrodes, the activities and current outputs of the covalently immobilized laccases were twice higher than the physically adsorbed ones. The sensitivity of these biocompatible bioelectrodes was evaluated by measuring their catalytic efficiency for oxygen reduction in the presence of ABTS as non-phenolic redox substrate and 2,6-dimethoxyphenol (DMP) as phenolic one. Sensitivities of around 4.8 μA mg{sup −1} L and 2.7 μA mg{sup −1} L were attained for ABTS and DMP respectively. An excellent stability of this laccase biosensor was observed for over 6 months. - Highlights: • Low pressure plasma was used to generate stable allylamine coating. • Laccase from Trametes versicolor was physisorbed and covalently immobilized. • Best biosensor efficiency obtained for the covalently immobilized laccases • Sensitivities of 4.8 μA mg{sup −1} L and 2.7 μA mg{sup −1} L for ABTS and DMP respectively.

An overview of technologies for immobilization of enzymes and surface analysis techniques for immobilized enzymes

Science.gov (United States)

Mohamad, Nur Royhaila; Marzuki, Nur Haziqah Che; Buang, Nor Aziah; Huyop, Fahrul; Wahab, Roswanira Abdul

2015-01-01

The current demands of sustainable green methodologies have increased the use of enzymatic technology in industrial processes. Employment of enzyme as biocatalysts offers the benefits of mild reaction conditions, biodegradability and catalytic efficiency. The harsh conditions of industrial processes, however, increase propensity of enzyme destabilization, shortening their industrial lifespan. Consequently, the technology of enzyme immobilization provides an effective means to circumvent these concerns by enhancing enzyme catalytic properties and also simplify downstream processing and improve operational stability. There are several techniques used to immobilize the enzymes onto supports which range from reversible physical adsorption and ionic linkages, to the irreversible stable covalent bonds. Such techniques produce immobilized enzymes of varying stability due to changes in the surface microenvironment and degree of multipoint attachment. Hence, it is mandatory to obtain information about the structure of the enzyme protein following interaction with the support surface as well as interactions of the enzymes with other proteins. Characterization technologies at the nanoscale level to study enzymes immobilized on surfaces are crucial to obtain valuable qualitative and quantitative information, including morphological visualization of the immobilized enzymes. These technologies are pertinent to assess efficacy of an immobilization technique and development of future enzyme immobilization strategies. PMID:26019635

Direct electrochemistry of horseradish peroxidase immobilized on electrografted 4-ethynylphenyl film via click chemistry

International Nuclear Information System (INIS)

Ran Qin; Peng Ru; Liang Cong; Ye Siqiu; Xian Yuezhong; Zhang Wenjing; Jin Litong

2011-01-01

Graphical abstract: Hydrogen peroxide biosensor was developed based on electrochemically assisted aryldiazonium salt chemistry and click chemistry. Highlights: → A simple, versatile two-step approach, which is based on electrochemically assisted aryldiazonium salt chemistry and Cu(I)-catalyzed azide alkyne cycloaddition (CuAAC) reaction has been developed for covalent redox proteins immobilization and biosensing for the first time. In this work, azido group modified HRP was covalently grafted on 4-ethylnylphenyl diazonium compound via CuAAC reaction and a novel electrochemical hydrogen peroxide biosensor was successfully fabricated. - Abstract: In this paper, a simple two-step approach for redox protein immobilization was introduced. Firstly, alkynyl-terminated film was formed on electrode surface by electrochemical reduction of 4-ethylnylphenyl (4-EP) diazonium compound. Then, horseradish peroxidase (HRP) modified with azido group was covalently immobilized onto the electrografted film via click reaction. Reflection absorption infrared (RAIR) spectroscopy and electrochemical methods were used to characterize the modification process. The results indicate that HRP retains its native structure and shows fast direct electron transfer. Moreover, the immobilized HRP shows excellent electrocatalytic reduction activity toward H 2 O 2 with a linear range of 5.0 x 10 -6 to 9.3 x 10 -4 mol L -1 .

Direct electrochemistry of horseradish peroxidase immobilized on electrografted 4-ethynylphenyl film via click chemistry

Energy Technology Data Exchange (ETDEWEB)

Ran Qin; Peng Ru; Liang Cong; Ye Siqiu [Department of Chemistry, East China Normal University, Shanghai 200062 (China); Xian Yuezhong, E-mail: yzxian@chem.ecnu.edu.cn [Department of Chemistry, East China Normal University, Shanghai 200062 (China); Zhang Wenjing; Jin Litong [Department of Chemistry, East China Normal University, Shanghai 200062 (China)

2011-07-04

Graphical abstract: Hydrogen peroxide biosensor was developed based on electrochemically assisted aryldiazonium salt chemistry and click chemistry. Highlights: > A simple, versatile two-step approach, which is based on electrochemically assisted aryldiazonium salt chemistry and Cu(I)-catalyzed azide alkyne cycloaddition (CuAAC) reaction has been developed for covalent redox proteins immobilization and biosensing for the first time. In this work, azido group modified HRP was covalently grafted on 4-ethylnylphenyl diazonium compound via CuAAC reaction and a novel electrochemical hydrogen peroxide biosensor was successfully fabricated. - Abstract: In this paper, a simple two-step approach for redox protein immobilization was introduced. Firstly, alkynyl-terminated film was formed on electrode surface by electrochemical reduction of 4-ethylnylphenyl (4-EP) diazonium compound. Then, horseradish peroxidase (HRP) modified with azido group was covalently immobilized onto the electrografted film via click reaction. Reflection absorption infrared (RAIR) spectroscopy and electrochemical methods were used to characterize the modification process. The results indicate that HRP retains its native structure and shows fast direct electron transfer. Moreover, the immobilized HRP shows excellent electrocatalytic reduction activity toward H{sub 2}O{sub 2} with a linear range of 5.0 x 10{sup -6} to 9.3 x 10{sup -4} mol L{sup -1}.

Influence of acetylcholinesterase immobilization on the photoluminescence properties of mesoporous silicon surface

Energy Technology Data Exchange (ETDEWEB)

Saleem, Muhammad [Department of Chemistry, Kongju National University, Gongju, Chungnam 314-701 (Korea, Republic of); Rafiq, Muhammad; Seo, Sung-Yum [Department of Biology, Kongju National University, Gongju, Chungnam 314-701 (Korea, Republic of); Lee, Ki Hwan, E-mail: khlee@kongju.ac.kr [Department of Chemistry, Kongju National University, Gongju, Chungnam 314-701 (Korea, Republic of)

2014-07-01

Acetylcholinesterase immobilized p-type porous silicon surface was prepared by covalent attachment. The immobilization procedure was based on support surface chemical oxidation, silanization, surface activation with cyanuric chloride and finally covalent attachment of free enzyme on the cyanuric chloride activated porous silicon surface. Different pore diameter of porous silicon samples were prepared by electrochemical etching in HF based electrolyte solution and appropriate sample was selected suitable for enzyme immobilization with maximum trapping ability. The surface modification was studied through field emission scanning electron microscope, EDS, FT-IR analysis, and photoluminescence measurement by utilizing the fluctuation in the photoluminescence of virgin and enzyme immobilized porous silicon surface. Porous silicon showed strong photoluminescence with maximum emission at 643 nm and immobilization of acetylcholinesterase on porous silicon surface cause considerable increment on the photoluminescence of porous silicon material while acetylcholinesterase free counterpart did not exhibit any fluorescence in the range of 635–670 nm. The activities of the free and immobilized enzymes were evaluated by spectrophotometric method by using neostigmine methylsulfate as standard enzyme inhibitor. The immobilized enzyme exhibited considerable response toward neostigmine methylsulfate in a dose dependent manner comparable with that of its free counterpart alongside enhanced stability, easy separation from the reaction media and significant saving of enzyme. It was believed that immobilized enzyme can be exploited in organic and biomolecule synthesis possessing technical and economical prestige over free enzyme and prominence of easy separation from the reaction mixture.

Influence of acetylcholinesterase immobilization on the photoluminescence properties of mesoporous silicon surface

International Nuclear Information System (INIS)

Saleem, Muhammad; Rafiq, Muhammad; Seo, Sung-Yum; Lee, Ki Hwan

2014-01-01

Acetylcholinesterase immobilized p-type porous silicon surface was prepared by covalent attachment. The immobilization procedure was based on support surface chemical oxidation, silanization, surface activation with cyanuric chloride and finally covalent attachment of free enzyme on the cyanuric chloride activated porous silicon surface. Different pore diameter of porous silicon samples were prepared by electrochemical etching in HF based electrolyte solution and appropriate sample was selected suitable for enzyme immobilization with maximum trapping ability. The surface modification was studied through field emission scanning electron microscope, EDS, FT-IR analysis, and photoluminescence measurement by utilizing the fluctuation in the photoluminescence of virgin and enzyme immobilized porous silicon surface. Porous silicon showed strong photoluminescence with maximum emission at 643 nm and immobilization of acetylcholinesterase on porous silicon surface cause considerable increment on the photoluminescence of porous silicon material while acetylcholinesterase free counterpart did not exhibit any fluorescence in the range of 635–670 nm. The activities of the free and immobilized enzymes were evaluated by spectrophotometric method by using neostigmine methylsulfate as standard enzyme inhibitor. The immobilized enzyme exhibited considerable response toward neostigmine methylsulfate in a dose dependent manner comparable with that of its free counterpart alongside enhanced stability, easy separation from the reaction media and significant saving of enzyme. It was believed that immobilized enzyme can be exploited in organic and biomolecule synthesis possessing technical and economical prestige over free enzyme and prominence of easy separation from the reaction mixture.

Preparation of Laccase Immobilized Cryogels and Usage for Decolorization

Directory of Open Access Journals (Sweden)

Murat Uygun

2013-01-01

Full Text Available Poly(methyl methacrylate-co-glycidyl methacrylate (poly(MMA-co-GMA cryogels were synthesized by radical cryopolymerization technique. Then, laccase enzyme was covalently attached to the cryogel and characterized by using swelling studies and SEM and EDX analyses. Kinetic properties and optimum conditions of the immobilized and free laccase were studied and it was found that of the immobilized laccase was lower than that of free laccase. of the immobilized laccase was increased upon immobilization. Optimum pH was found to be 4.0 for each type of laccase, while optimum temperature was shifted to the warmer region after the immobilization. It was also found that thermal stability of the immobilized laccase was higher than that of free laccase. Immobilized laccase could be used for 10 times successive reuse with no significant decrease in its activity. Also, these laccase immobilized cryogels were successfully used for the decolorization of seven different dyes.

Different Covalent Immobilizations Modulate Lipase Activities of Hypocrea pseudokoningii

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Marita G. Pereira

2017-09-01

Full Text Available Enzyme immobilization can promote several advantages for their industrial application. In this work, a lipase from Hypocrea pseudokoningii was efficiently linked to four chemical supports: agarose activated with cyanogen bromide (CNBr, glyoxyl-agarose (GX, MANAE-agarose activated with glutaraldehyde (GA and GA-crosslinked with glutaraldehyde. Results showed a more stable lipase with both the GA-crosslinked and GA derivatives, compared to the control (CNBr, at 50 °C, 60 °C and 70 °C. Moreover, all derivatives were stabilized when incubated with organic solvents at 50%, such as ethanol, methanol, n-propanol and cyclohexane. Furthermore, lipase was highly activated (4-fold in the presence of cyclohexane. GA-crosslinked and GA derivatives were more stable than the CNBr one in the presence of organic solvents. All derivatives were able to hydrolyze sardine, açaí (Euterpe oleracea, cotton seed and grape seed oils. However, during the hydrolysis of sardine oil, GX derivative showed to be 2.3-fold more selectivity (eicosapentaenoic acid (EPA/docosahexaenoic acid (DHA ratio than the control. Additionally, the types of immobilization interfered with the lipase enantiomeric preference. Unlike the control, the other three derivatives preferably hydrolyzed the R-isomer of 2-hydroxy-4-phenylbutanoic acid ethyl ester and the S-isomer of 1-phenylethanol acetate racemic mixtures. On the other hand, GX and CNBr derivatives preferably hydrolyzed the S-isomer of butyryl-2-phenylacetic acid racemic mixture while the GA and GA-crosslink derivatives preferably hydrolyzed the R-isomer. However, all derivatives, including the control, preferably hydrolyzed the methyl mandelate S-isomer. Moreover, the derivatives could be used for eight consecutive cycles retaining more than 50% of their residual activity. This work shows the importance of immobilization as a tool to increase the lipase stability to temperature and organic solvents, thus enabling the possibility of

Biocatalytic Behaviour of Immobilized Rhizopus oryzae Lipase in the 1,3-Selective Ethanolysis of Sunflower Oil to Obtain a Biofuel Similar to Biodiesel

Directory of Open Access Journals (Sweden)

Carlos Luna

2014-08-01

Full Text Available A new biofuel similar to biodiesel was obtained in the 1,3-selective transesterification reaction of sunflower oil with ethanol using as biocatalyst a Rhizopus oryzae lipase (ROL immobilized on Sepiolite, an inorganic support. The studied lipase was a low cost powdered enzyme preparation, Biolipase-R, from Biocon-Spain, a multipurpose additive used in food industry. In this respect, it is developed a study to optimize the immobilization procedure of these lipases on Sepiolite. Covalent immobilization was achieved by the development of an inorganic-organic hybrid linker formed by a functionalized hydrocarbon chain with a pendant benzaldehyde, bonded to the AlPO4 support surface. Thus, the covalent immobilization of lipases on amorphous AlPO4/sepiolite (20/80 wt % support was evaluated by using two different linkers (p-hydroxybenzaldehyde and benzylamine-terephthalic aldehyde, respectively. Besides, the catalytic behavior of lipases after physical adsorption on the demineralized sepiolite  was also evaluated. Obtained results indicated that covalent immobilization with the p-hydroxybenzaldehyde linker gave the best biocatalytic behavior. Thus, this covalently immobilized lipase showed a remarkable stability as well as an excellent capacity of reutilization (more than five successive reuses without a significant loss of its initial catalytic activity. This could allow a more efficient fabrication of biodiesel minimizing the glycerol waste production.

Enzymatic removal of phenol and p-chlorophenol in enzyme reactor: Horseradish peroxidase immobilized on magnetic beads

International Nuclear Information System (INIS)

Bayramoglu, Guelay; Arica, M. Yakup

2008-01-01

Horseradish peroxidase was immobilized on the magnetic poly(glycidylmethacrylate-co-methylmethacrylate) (poly(GMA-MMA)), via covalent bonding and used for the treatment of phenolic wastewater in continuous systems. For this purposes, horseradish peroxidase (HRP) was covalently immobilized onto magnetic poly(GMA-MMA) beds using glutaraldehyde (GA) as a coupling agent. The maximum HRP immobilization capacity of the magnetic poly(GMA-MMA)-GA beads was 3.35 mg g -1 . The immobilized HRP retained 79% of the activity of the free HRP used for immobilization. The immobilized HRP was used for the removal of phenol and p-chlorophenol via polymerization of dissolved phenols in the presence of hydrogen peroxide (H 2 O 2 ). The effect of pH and temperature on the phenol oxidation rate was investigated. The results were compared with the free HRP, which showed that the optimum pH value for the immobilized HRP is similar to that for the free HRP. The optimum pH value for free and immobilized HRP was observed at pH 7.0. The optimum temperature for phenols oxidation with immobilized HRP was between 25 and 35 deg. C and the immobilized HRP has more resistance to temperature inactivation than that of the free form. Finally, the immobilized HRP was operated in a magnetically stabilized fluidized bed reactor, and phenols were successfully removed in the enzyme reactor

Quantitative analysis of immobilized penicillinase using enzyme-modified AlGaN/GaN field-effect transistors.

Science.gov (United States)

Müntze, Gesche Mareike; Baur, Barbara; Schäfer, Wladimir; Sasse, Alexander; Howgate, John; Röth, Kai; Eickhoff, Martin

2015-02-15

Penicillinase-modified AlGaN/GaN field-effect transistors (PenFETs) are utilized to systematically investigate the covalently immobilized enzyme penicillinase under different experimental conditions. We demonstrate quantitative evaluation of covalently immobilized penicillinase layers on pH-sensitive field-effect transistors (FETs) using an analytical kinetic PenFET model. This kinetic model is explicitly suited for devices with thin enzyme layers that are not diffusion-limited, as it is the case for the PenFETs discussed here. By means of the kinetic model it was possible to extract the Michaelis constant of covalently immobilized penicillinase as well as relative transport coefficients of the different species associated with the enzymatic reaction which, exempli gratia, give information about the permeability of the enzymatic layer. Based on this analysis we quantify the reproducibility and the stability of the analyzed PenFETs over the course of 33 days as well as the influence of pH and buffer concentration on the properties of the enzymatic layer. Thereby the stability measurements reveal a Michalis constant KM of (67 ± 13)μM while the chronological development of the relative transport coefficients suggests a detachment of physisorbed penicillinase during the first two weeks since production. Our results show that AlGaN/GaN PenFETs prepared by covalent immobilization of a penicillinase enzyme layer present a powerful tool for quantitative analysis of enzyme functionality. Copyright © 2014 Elsevier B.V. All rights reserved.

Immobilization of 2,4- and 2,6-Dinitrotoluenes in Soils and Compost

National Research Council Canada - National Science Library

Pennington, Judith

2003-01-01

Covalent bonding of amino transformation products of trinitrotoluene (TNT) to functional groups on humic acid results in immobilized products that are not hydrolyzable, microbially degradable, or leachable...

Immobilization of indigenous holocellulase on iron oxide (Fe2O3) nanoparticles enhanced hydrolysis of alkali pretreated paddy straw.

Science.gov (United States)

Kumar, Ajay; Singh, Surender; Tiwari, Rameshwar; Goel, Renu; Nain, Lata

2017-03-01

The holocellulase from Aspergillus niger SH3 was characterized and found to contain 125 proteins including cellulases (26), hemicellulases (21), chitinases (10), esterases (6), amylases (4) and hypothetical protein (32). The crude enzyme was immobilized on five different nanoparticles (NPs) via physical adsorption and covalent coupling methods. The enzyme-nanoparticle complexes (ENC) were screened for protein binding, enzymatic activities and immobilization efficiency. Magnetic enzyme-nanoparticle complexes (MENC) showed higher immobilization efficiency (60-80%) for most of the enzymes. MENC also showed better catalytic efficiencies in term of higher V max and lower K m than free enzyme. Saccharification yields from alkali treated paddy straw were higher (375.39mg/gds) for covalently immobilized MENC than free enzyme (339.99mg/gds). The immobilized enzyme was used for two cycles of saccharification with 55% enzyme recovery. Hence, this study for the first time demonstrated the immobilization of indigenous enzyme and its utilization for saccharification of paddy straw. Copyright © 2016 Elsevier B.V. All rights reserved.

Immobilization of inorganic pyrophosphatase on nanodiamond particles retaining its high enzymatic activity.

Science.gov (United States)

Rodina, Elena V; Valueva, Anastasiya V; Yakovlev, Ruslan Yu; Vorobyeva, Nataliya N; Kulakova, Inna I; Lisichkin, Georgy V; Leonidov, Nikolay B

2015-12-21

Nanodiamond (ND) particles are popular platforms for the immobilization of molecular species. In the present research, enzyme Escherichia coli inorganic pyrophosphatase (PPase) was immobilized on detonation ND through covalent or noncovalent bonding and its enzymatic activity was characterized. Factors affecting adsorption of PPase such as ND size and surface chemistry were studied. The obtained material is a submicron size association of ND particles and protein molecules in approximately equal amounts. Both covalently and noncovalently immobilized PPase retains a significant enzymatic activity (up to 95% of its soluble form) as well as thermostability. The obtained hybrid material has a very high enzyme loading capacity (∼1 mg mg(-1)) and may be considered as a promising delivery system of biologically active proteinaceous substances, particularly in the treatment of diseases such as calcium pyrophosphate crystal deposition disease and related pathologies. They can also be used as recoverable heterogeneous catalysts in the traditional uses of PPase.

The effect of the shape of single, sub-ms voltage pulses on the rates of surface immobilization and hybridization of DNA

International Nuclear Information System (INIS)

Cabeca, R; Rodrigues, M; Chu, V; Conde, J P; Prazeres, D M F

2009-01-01

Electric fields generated by single square and sinusoidal voltage pulses with amplitudes below 2 V were used to assist the covalent immobilization of single-stranded, thiolated DNA probes, onto a chemically functionalized SiO 2 surface and to assist the specific hybridization of single-stranded DNA targets with immobilized complementary probes. The single-stranded immobilized DNA probes were either covalently immobilized (chemisorption) or electrostatically adsorbed (physisorption) to a chemically functionalized surface. Comparing the speed of electric field assisted immobilization and hybridization with the corresponding control reactions (without electric field), an increase of several orders of magnitude is observed, with the reaction timescaled down from 1 to 2 h to a range between 100 ns and 1 ms. The influence of the shape of the voltage pulse (square versus sinusoidal) and its duration were studied for both immobilization and hybridization reactions. The results show that pulsed electric fields are a useful tool to achieve temporal and spatial control of surface immobilization and hybridization reactions of DNA.

Immobilization of catalase on chitosan and amino acid- modified chitosan beads.

Science.gov (United States)

Başak, Esra; Aydemir, Tülin

2013-08-01

Bovine liver catalase was covalently immobilized onto amino acid-modified chitosan beads. The beads were characterized with SEM, FTIR, TGA and the effects of immobilization on optimum pH and temperature, thermostability, reusability were evaluated. Immobilized catalase showed the maximal enzyme activity at pH 7.0 at 30°C. The kinetic parameters, Km and Vmax, for immobilized catalase on alanine-chitosan beads and lysine-chitosan beads were estimated to be 25.67 mM, 27 mM and 201.39 μmol H2O2/min, 197.50 μmol H2O2/min, respectively. The activity of the immobilized catalase on Ala-CB and Lys-CB retained 40% of its high initial activity after 100 times of reuse.

Optimizing Immobilized Enzyme Performance in Cell-Free Environments to Produce Liquid Fuels

Energy Technology Data Exchange (ETDEWEB)

Belfort, Georges [Rensselaer Polytechnic Inst., Troy, NY (United States). Dept. of Chemical and Biological Engineering; Grimaldi, Joseph J. [Rensselaer Polytechnic Inst., Troy, NY (United States). Dept. of Chemical and Biological Engineering

2015-01-27

Limitations on biofuel production using cell culture (Escherichia coli, Clostridium, Saccharomyces cerevisiae, brown microalgae, blue-green algae and others) include low product (alcohol) concentrations (≤0.2 vol%) due to feedback inhibition, instability of cells, and lack of economical product recovery processes. To overcome these challenges, an alternate simplified biofuel production scheme was tested based on a cell-free immobilized enzyme system. Using this cell free system, we were able to obtain about 2.6 times higher concentrations of iso-butanol using our non-optimized system as compared with live cell systems. This process involved two steps: (i) converts acid to aldehyde using keto-acid decarboxylase (KdcA), and (ii) produces alcohol from aldehyde using alcohol dehydrogenase (ADH) with a cofactor (NADH) conversion from inexpensive formate using a third enzyme, formate dehydrogenase (FDH). To increase stability and conversion efficiency with easy separations, the first two enzymes were immobilized onto methacrylate resin. Fusion proteins of labile KdcA (fKdcA) were expressed to stabilize the covalently immobilized KdcA. Covalently immobilized ADH exhibited long-term stability and efficient conversion of aldehyde to alcohol over multiple batch cycles without fusions. High conversion rates and low protein leaching were achieved by covalent immobilization of enzymes on methacrylate resin. The complete reaction scheme was demonstrated by immobilizing both ADH and fKdcA and using FDH free in solution. The new system without in situ removal of isobutanol achieved a 55% conversion of ketoisovaleric acid to isobutanol at a concentration of 0.5 % (v/v). Further increases in titer will require continuous removal of the isobutanol using our novel brush membrane system that exhibits a 1.5 fold increase in the separation factor of isobutanol from water versus that obtained for commercial silicone rubber membranes. These bio-inspired brush membranes are based on the

Horseradish peroxidase and toluidine blue covalently immobilized leak-free sol-gel composite biosensor for hydrogen peroxide.

Science.gov (United States)

Thenmozhi, K; Narayanan, S Sriman

2017-01-01

The enzyme horseradish peroxidase and the water-soluble mediator toluidine blue were covalently immobilized to 3-aminopropyl trimethoxy silane precursor through glutaraldehyde crosslinker. A rigid ceramic composite electrode was fabricated from this modified silane along with graphite powder, which resulted in an amperometric biosensor for H 2 O 2 . The electrochemical behaviour of the modified biosensor was monitored using cyclic voltammetry in the potential range of 0.2V to -0.4V vs SCE. The biosensor exhibited a stable voltammogram with cathodic peak at -0.234V and anodic peak at -0.172V, with a formal potential of -0.203V. Various factors influencing the performance of the biosensor such as buffer solution, pH, temperature and potential were examined for optimizing the working conditions. The modified biosensor exhibited a good catalytic behaviour for the reduction of H 2 O 2 at a lower potential of -0.25V without any barrier from possible interferents. The analytical working range was found to be 0.429μM to 0.455mM of H 2 O 2 with a detection limit of 0.171μM. The fabricated biosensor is robust for long-term usage in addition to the high sensitivity, rapid response and having an advantage of surface renewability by simple mechanical polishing. Copyright © 2016 Elsevier B.V. All rights reserved.

Covalent growth factor tethering to direct neural stem cell differentiation and self-organization.

Science.gov (United States)

Ham, Trevor R; Farrag, Mahmoud; Leipzig, Nic D

2017-04-15

Tethered growth factors offer exciting new possibilities for guiding stem cell behavior. However, many of the current methods present substantial drawbacks which can limit their application and confound results. In this work, we developed a new method for the site-specific covalent immobilization of azide-tagged growth factors and investigated its utility in a model system for guiding neural stem cell (NSC) behavior. An engineered interferon-γ (IFN-γ) fusion protein was tagged with an N-terminal azide group, and immobilized to two different dibenzocyclooctyne-functionalized biomimetic polysaccharides (chitosan and hyaluronan). We successfully immobilized azide-tagged IFN-γ under a wide variety of reaction conditions, both in solution and to bulk hydrogels. To understand the interplay between surface chemistry and protein immobilization, we cultured primary rat NSCs on both materials and showed pronounced biological effects. Expectedly, immobilized IFN-γ increased neuronal differentiation on both materials. Expression of other lineage markers varied depending on the material, suggesting that the interplay of surface chemistry and protein immobilization plays a large role in nuanced cell behavior. We also investigated the bioactivity of immobilized IFN-γ in a 3D environment in vivo and found that it sparked the robust formation of neural tube-like structures from encapsulated NSCs. These findings support a wide range of potential uses for this approach and provide further evidence that adult NSCs are capable of self-organization when exposed to the proper microenvironment. For stem cells to be used effectively in regenerative medicine applications, they must be provided with the appropriate cues and microenvironment so that they integrate with existing tissue. This study explores a new method for guiding stem cell behavior: covalent growth factor tethering. We found that adding an N-terminal azide-tag to interferon-γ enabled stable and robust Cu-free 'click

Magnetically modified bacterial cellulose: A promising carrier for immobilization of affinity ligands, enzymes, and cells

Energy Technology Data Exchange (ETDEWEB)

Baldikova, Eva [Global Change Research Institute, CAS, Na Sadkach 7, 370 05 Ceske Budejovice (Czech Republic); Pospiskova, Kristyna [Regional Centre of Advanced Technologies and Materials, Palacky University, Slechtitelu 27, 783 71 Olomouc (Czech Republic); Ladakis, Dimitrios; Kookos, Ioannis K. [Department of Chemical Engineering, University of Patras, 26504 Patras, Rio (Greece); Koutinas, Apostolis A. [Department of Food Science and Human Nutrition, Agricultural University of Athens, Iera Odos 75, Athens 11855 (Greece); Safarikova, Mirka [Global Change Research Institute, CAS, Na Sadkach 7, 370 05 Ceske Budejovice (Czech Republic); Department of Nanobiotechnology, Biology Centre, ISB, CAS, Na Sadkach 7, 370 05 Ceske Budejovice (Czech Republic); Safarik, Ivo, E-mail: safarik@nh.cas.cz [Global Change Research Institute, CAS, Na Sadkach 7, 370 05 Ceske Budejovice (Czech Republic); Regional Centre of Advanced Technologies and Materials, Palacky University, Slechtitelu 27, 783 71 Olomouc (Czech Republic); Department of Nanobiotechnology, Biology Centre, ISB, CAS, Na Sadkach 7, 370 05 Ceske Budejovice (Czech Republic)

2017-02-01

Bacterial cellulose (BC) produced by Komagataeibacter sucrofermentans was magnetically modified using perchloric acid stabilized magnetic fluid. Magnetic bacterial cellulose (MBC) was used as a carrier for the immobilization of affinity ligands, enzymes and cells. MBC with immobilized reactive copper phthalocyanine dye was an efficient adsorbent for crystal violet removal; the maximum adsorption capacity was 388 mg/g. Kinetic and thermodynamic parameters were also determined. Model biocatalysts, namely bovine pancreas trypsin and Saccharomyces cerevisiae cells were immobilized on MBC using several strategies including adsorption with subsequent cross-linking with glutaraldehyde and covalent binding on previously activated MBC using sodium periodate or 1,4-butanediol diglycidyl ether. Immobilized yeast cells retained approximately 90% of their initial activity after 6 repeated cycles of sucrose solution hydrolysis. Trypsin covalently bound after MBC periodate activation was very stable during operational stability testing; it could be repeatedly used for ten cycles of low molecular weight substrate hydrolysis without loss of its initial activity. - Highlights: • Bacterial cellulose was magnetically modified with magnetic fluid. • Magnetic cellulose is an efficient carrier for affinity ligands. • Enzymes and cells can be efficiently immobilized to magnetic cellulose.

Self-assembled monolayers of 1-alkenes on oxidized platinum surfaces as platforms for immobilized enzymes for biosensing

International Nuclear Information System (INIS)

Alonso, Jose Maria; Bielen, Abraham A.M.; Olthuis, Wouter; Kengen, Servé W.M.; Zuilhof, Han; Franssen, Maurice C.R.

2016-01-01

Highlights: • Three different oxidases are covalently attached to alkene based SAMs on PtOx. • Attached enzymes remain active and their activity is assessed by chronoamperometry. • Functionalized PtOx allows electron mediator free chronoamperometry measurements. • The thus formed enzyme electrodes are useful as biosensors for glucose and lactate. • Immobilization of human HAOX foresees in vivo lactate monitoring in humans. - Abstract: Alkene-based self-assembled monolayers grafted on oxidized Pt surfaces were used as a scaffold to covalently immobilize oxidase enzymes, with the aim to develop an amperometric biosensor platform. NH_2-terminated organic layers were functionalized with either aldehyde (CHO) or N-hydroxysuccinimide (NHS) ester-derived groups, to provide anchoring points for enzyme immobilization. The functionalized Pt surfaces were characterized by X-ray photoelectron spectroscopy (XPS), static water contact angle (CA), infrared reflection absorption spectroscopy (IRRAS) and atomic force microscopy (AFM). Glucose oxidase (GOX) was covalently attached to the functionalized Pt electrodes, either with or without additional glutaraldehyde crosslinking. The responses of the acquired sensors to glucose concentrations ranging from 0.5 to 100 mM were monitored by chronoamperometry. Furthermore, lactate oxidase (LOX) and human hydroxyacid oxidase (HAOX) were successfully immobilized onto the PtOx surface platform. The performance of the resulting lactate sensors was investigated for lactate concentrations ranging from 0.05 to 20 mM. The successful attachment of active enzymes (GOX, LOX and HAOX) on Pt electrodes demonstrates that covalently functionalized PtOx surfaces provide a universal platform for the development of oxidase enzyme-based sensors.

Self-assembled monolayers of 1-alkenes on oxidized platinum surfaces as platforms for immobilized enzymes for biosensing

Energy Technology Data Exchange (ETDEWEB)

Alonso, Jose Maria; Bielen, Abraham A.M. [Laboratory of Organic Chemistry, Wageningen University, Dreijenplein 8, 6703 HB, Wageningen (Netherlands); Olthuis, Wouter [BIOS Lab on a Chip Group, MESA+ and MIRA Institutes, University of Twente, P.O. Box 217, 7500 AE Enschede (Netherlands); Kengen, Servé W.M. [Laboratory of Microbiology, Wageningen University, 6703HB Wageningen (Netherlands); Zuilhof, Han, E-mail: han.zuilhof@wur.nl [Laboratory of Organic Chemistry, Wageningen University, Dreijenplein 8, 6703 HB, Wageningen (Netherlands); Department of Chemical and Materials Engineering, King Abdulaziz University, Jeddah 22254 (Saudi Arabia); Franssen, Maurice C.R., E-mail: maurice.franssen@wur.nl [Laboratory of Organic Chemistry, Wageningen University, Dreijenplein 8, 6703 HB, Wageningen (Netherlands)

2016-10-15

Highlights: • Three different oxidases are covalently attached to alkene based SAMs on PtOx. • Attached enzymes remain active and their activity is assessed by chronoamperometry. • Functionalized PtOx allows electron mediator free chronoamperometry measurements. • The thus formed enzyme electrodes are useful as biosensors for glucose and lactate. • Immobilization of human HAOX foresees in vivo lactate monitoring in humans. - Abstract: Alkene-based self-assembled monolayers grafted on oxidized Pt surfaces were used as a scaffold to covalently immobilize oxidase enzymes, with the aim to develop an amperometric biosensor platform. NH{sub 2}-terminated organic layers were functionalized with either aldehyde (CHO) or N-hydroxysuccinimide (NHS) ester-derived groups, to provide anchoring points for enzyme immobilization. The functionalized Pt surfaces were characterized by X-ray photoelectron spectroscopy (XPS), static water contact angle (CA), infrared reflection absorption spectroscopy (IRRAS) and atomic force microscopy (AFM). Glucose oxidase (GOX) was covalently attached to the functionalized Pt electrodes, either with or without additional glutaraldehyde crosslinking. The responses of the acquired sensors to glucose concentrations ranging from 0.5 to 100 mM were monitored by chronoamperometry. Furthermore, lactate oxidase (LOX) and human hydroxyacid oxidase (HAOX) were successfully immobilized onto the PtOx surface platform. The performance of the resulting lactate sensors was investigated for lactate concentrations ranging from 0.05 to 20 mM. The successful attachment of active enzymes (GOX, LOX and HAOX) on Pt electrodes demonstrates that covalently functionalized PtOx surfaces provide a universal platform for the development of oxidase enzyme-based sensors.

Stabilization of 5A1 urease by covalent attachement to wool | Ahmed ...

African Journals Online (AJOL)

The investigation of five bacterial strains for urease production referred that Bacillus licheniformis 5A1 had the highest urease activity (10.3U/ml/min) after 24h. The enzyme was covalently coupled to different carriers via glutaraldehyde, and wool gave the highest immobilization yield (76.4%) and retained 85% of the original ...

Immobilized enzymes in blood plasma exchangers via radiation grafting

Science.gov (United States)

Gombotz, Wayne; Hoffman, Allan; Schmer, Gottfried; Uenoyama, Satoshi

The enzyme asparaginase was immobilized onto a porous hollow polypropylene (PP) fiber blood plasma exchange device for the treatment of acute lymphocytic leukemia. The devices were first radiation grafted with polymethacrylic acid (poly(MAAc)). This introduces carboxyl groups onto the surface of the fibers. Several variables were studied in the grafting reaction including the effects of solvent type and monomer concentration. The carboxyl groups were activated with N-hydroxy succinimide (NHS) using carbodiimide chemistry. Asparaginase was then covalently immobilized on the activated surfaces. Quantitative relationships were found relating the percent graft to the amount of immobilized enzyme which was active. The enzyme reactor was tested both in vitro and in vivo using a sheep as an animal model.

Novel immobilizations of an adhesion peptide on the TiO2 surface: An XPS investigation

International Nuclear Information System (INIS)

Iucci, G.; Dettin, M.; Battocchio, C.; Gambaretto, R.; Bello, C. Di; Polzonetti, G.

2007-01-01

The covalent attachment of an adhesive peptide, reproducing the 351-359 sequence of human vitronectin, to oxidized titanium surfaces was investigated by XPS spectroscopy. The peptide enhances osteoblast adhesion to titanium, the most used biomaterial for implants and prostheses. Core level spectra of the TiO 2 surface and of the biomimetic surface were investigated. Novel selective covalent immobilization of (351-359) HVP was carried out by treatment of the TiO 2 surface with (3-aminopropyl) triethoxysilane, glutaric anhydride and a side chain protected peptide sequence presenting only a free terminal amino group, followed by side chain deprotection. An alternative strategy for covalent attachment consists in photoactivation of physisorbed (351-359) HVP directly on the TiO 2 surface; samples were incubated with HVP solution and subsequently irradiated with UV light. A comparison with the results previously obtained for non-selective HVP immobilization will be discussed

Acyclic N-halamine-immobilized polyurethane: Preparation and antimicrobial and biofilm-controlling functions

Science.gov (United States)

Luo, Jie; Porteous, Nuala; Lin, Jiajin; Sun, Yuyu

2015-01-01

Hydroxyl groups were introduced onto polyurethane surfaces through 1,6-hexamethylene diisocyanate activation, followed by diethanolamine hydroxylation. Polymethacrylamide was covalently attached to the hydroxylated polyurethane through surface grafting polymerization of methacrylamide using cerium (IV) ammonium nitrate as an initiator. After bleach treatment, the amide groups of the covalently bound polymethacrylamide chains were transformed into N-halamines. The new N-halamine-immobilized polyurethane provided a total sacrifice of 107–108 colony forming units per milliliter of Staphylococcus aureus (Gram-positive bacteria), Escherichia coli (Gram-negative bacteria), and Candida albicans (fungi) within 10 min and successfully prevented bacterial and fungal biofilm formation. The antimicrobial and biofilm-controlling effects were both durable and rechargeable, pointing to great potentials of the new acyclic N-halamine-immobilized polyurethane for a broad range of related applications. PMID:26089593

Improving Pullulanase Catalysis via Reversible Immobilization on Modified Fe3O4@Polydopamine Nanoparticles.

Science.gov (United States)

Wang, Jianfeng; Liu, Zhongmei; Zhou, Zhemin

2017-08-01

To improve the catalysis of pullulanase from Anoxybacillus sp.WB42, Fe 3 O 4 @polydopamine nanoparticles (Fe 3 O 4 @PDA) were prepared and modified with functional groups for immobilization of pullulanases via covalent binding or ionic adsorption. Immobilized pullulanases had lower thermal stability than that of free pullulanase, whereas their catalysis depended on the surface characteristics of nanoparticles. As for covalent immobilization of pullulanases onto Fe 3 O 4 @PDA derivatives, the spacer grafted onto Fe 3 O 4 @PDA made the catalytic efficiency of pullulanase increase up to the equivalence of free enzyme but dramatically reduced the pullulanase thermostability. In contrast, pullulanases bounded ionically to Fe 3 O 4 @PDA derivatives had higher activity recovery and catalytic efficiency, and their catalytic behaviors varied with the modifier grafted onto Fe 3 O 4 @PDA. Among these immobilized pullulanases, ionic adsorption of pullulanase on Fe 3 O 4 @PDA-polyethyleneimine-glycidyltrimethylammonium gave a high-performance and durable catalyst, which displayed not only 1.5-fold increase in catalytic efficiency compared to free enzyme but also a significant improvement in operation stability with a half of initial activity after 27 consecutive cycles with a total reaction time of 13.5 h, and was reversible, making this nanoparticle reusable for immobilization.

Effect of temperature and mixing speed on immobilization of crude enzyme from Aspergillus niger on chitosan for hydrolyzing cellulose

Science.gov (United States)

Hamzah, Afan; Gek Ela Kumala, P.; Ramadhani, Dwi; Maziyah, Nurul; Rahmah, Laila Nur; Soeprijanto, Widjaja, Arief

2017-05-01

Conversion of cellulose into reducing sugar through enzymatic hydrolysis has advantageous because it produces greater product yield, higher selectivity, require less energy, more moderate operating conditions and environment friendly. However, the nature of the enzyme that is difficult to separate and its expensive price become an obstacle. These obstacles can be overcome by immobilizing the enzyme on chitosan material so that the enzyme can be reused. Chitosan is chosen because it is cheap, inert, hydrophilic, and biocompatible. In this research, we use covalent attachment and combination between covalent attachment and cross-linking method for immobilizing crude enzyme. This research was focusing in study of Effect of temperature and mixing speed on Immobilization Enzyme From Aspergillus Niger on Chitosan For Hydrolyzing both soluble (Carboxymethylcellulose) and insoluble Cellulose (coconut husk). This Research was carried out by three main step. First, coconut husk was pre-treated mechanically and chemically, Second, Crude enzyme from Aspergillus niger strain was immobilized on chitosan in various immobilization condition. At last, the pre-treated coconut husk and Carboxymetylcellulose (CMC) were hydrolyzed by immobilized cellulose on chitosan for reducing sugar production. The result revealed that the most reducing sugar produced by immobilized enzyme on chitosan+GDA with immobilization condition at 30 °C and 125 rpm. Enzyme immobilized on chitosan cross-linked with GDA produced more reducing sugar from preteated coconut husk than enzyme immobilized on chitosan.

The influence of covalent immobilization conditions on antibody accessibility on nanoparticles

NARCIS (Netherlands)

Saha, Bedabrata; Songe, Pål; Evers, Toon H.; Prins, Menno W.J.

2017-01-01

The accessibility of particle-coupled antibodies is important for many analytical applications, but comprehensive data on parameters controlling the accessibility are scarce. Here we report on the site-specific accessibility of monoclonal antibodies, immobilized on magnetic nanoparticles (500 nm) by

Fabrication of molecular hybrid films of gold nanoparticle and polythiophene by covalent assembly

Energy Technology Data Exchange (ETDEWEB)

Sundaramurthy, Jayaraman, E-mail: jsu2@np.edu.sg [Department of Chemical & Biomolecular Engineering, National University of Singapore, Block E5, 4 Engineering Drive 4, 117576 (Singapore); Environmental & Water Technology Centre of Innovation, Ngee Ann Polytechnic, 599489 (Singapore); Dharmarajan, Rajarathnam [CERAR, University of South Australia, Mawson Lakes, SA 5095 (Australia); Srinivasan, M.P., E-mail: chesmp@nus.edu.sg [Department of Chemical & Biomolecular Engineering, National University of Singapore, Block E5, 4 Engineering Drive 4, 117576 (Singapore)

2015-08-31

This work demonstrates the fabrication of molecular hybrid films comprising gold nanoparticles (AuNPs) incorporated in covalently assembled, substituted polythiophene (poly(3-(2-bromoethoxy)ethoxymethylthiophene-2,5-diyl (PBrEEMT))) films by different surface chemistry routes. AuNPs are incorporated in the immobilized polythiophene matrix due to its affinity for amine and sulfur. The amount of AuNPs present depends on the nature of the incorporation, the extent of film coverage and interaction of thiophene and amine groups. PBrEEMT films functionalized with amine rich polyallylamine immobilize greater numbers of AuNPs due to more extensive gold–amine interactions. Covalent binding between AuNP and PBrEEMT films was accomplished by using pre-functionalised AuNPs (4-aminothiophenol functionalized AuNPs). Atomic force microscopy, field emission scanning electron microscopy and X-ray photoelectron spectroscopy were used to study the morphology and chemical constituents of assembled films. These approaches will pave the way for developing facile methods for nanoparticle incorporation and will also facilitate direct interaction of nanoparticles with the conducting polymer matrix and enhance the electrical properties of the films. - Highlights: • Covalent molecular assembly enabled the fabrication of molecular hybrid films. • Monomeric and polymeric species were employed as intermediate linkers. • Adopted approaches facilitated the direct interaction of gold nanoparticle in films. • The amount of nanoparticle incorporation depended on the extent of film coverage.

Magnetic Parkia pendula seed gum as matrix for Concanavalin A lectin immobilization and its application in affinity purification

Directory of Open Access Journals (Sweden)

MOACYR J.B.M. RÊGO

2014-09-01

Full Text Available The present work aimed to magnetize Parkia pendula seeds gum and use it as a matrix for Concanavalin A covalent immobilization. This composite was applied in affinity purification of glycoconjugates. Parkia pendula seeds were hydrated and the gum provenient from the supernatant was precipitated and washed with ethanol and dried. The gum was magnetized in co-precipitation using solutions of Fe+2 and Fe+3. Matrix activation was accomplished with NaIO4. Magnetized Parkia pendula seeds gum with covalently immobilized Concanavalin A was used as an affinity matrix for the recognition of bovine serum fetuin glycoprotein. Fetuin elution was carried out with a solution of glucose (300mM and evaluated through SDS-PAGE. The efficiency of lectin immobilization and fetuin purification were 63% and 14%, respectively. These results indicate that the composite produced is a promising magnetic polysaccharide matrix for lectins immobilization. Thus, such system can be applied for affinity purification allowing an easy recovery by magnetic field.

Immobilization of cellulase on functionalized cobalt ferrite nanoparticles

International Nuclear Information System (INIS)

Bohara, Raghvendra Ashok; Thorat, Nanasaheb Devappa; Pawar, Shivaji Hariba

2016-01-01

Amine functionalized cobalt ferrite (AF-CoFe 2 O 4 ) magnetic nanoparticles (MNPs) were used for immobilization of cellulase enzyme via 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDS) and N-hydroxysuccinimide (NHS) coupling reaction. The structural, morphological and magnetic properties of AF-CoFe 2 O 4 were determined. TEM micrograph revealed a mean diameter of -8 nm and showed that the AF-CoFe 2 O 4 remain distinct with no significant change in size after binding with cellulase. Fourier transform infrared (FT-IR) spectroscopy confirmed the binding of cellulase to AF-CoFe 2 O 4 . The properties of immobilized cellulase were investigated by optimizing binding efficiency, pH, temperature and reusability. The results showed that the immobilized cellulase has higher thermal stability than free cellulase, which might be due to covalent interaction between cellulase and AF-CoFe 2 O 4 surface. The immobilized cellulase also showed good reusability after recovery. Therefore, AF-CoFe 2 O 4 MNPs can be considered as promising candidate for enzyme immobilization.

MUCOADHESIVE GEL WITH IMMOBILIZED LYSOZYME: PREPARATION AND PROPERTIES

Directory of Open Access Journals (Sweden)

Dekina S. S.

2015-08-01

Full Text Available The study of non-covalent immobilized lysozyme, as well as physico-chemical and biochemical properties of obtained mucoadhesive gel was the aim of the research. Lysozyme activity was determined by bacteriolytic method (Micrococcus lysodeikticus cells acetone powder was a substrate. Lysozyme immobilization was conducted by the method of entrapment in gel. Enzyme carrier interaction was studied by viscometric, spectrophotometric and spectrofluorimetric methods. Mucoadhesive gel with immobilized lysozyme, possessing antiinflammatory and antimicrobial activities, was prepared. Due to immobilization, protein-polymer complex with the original enzymatic activity was formed. The product is characterized by high mucoadhesive properties, quantitative retaining of protein and bacteriolytic activity, prolonged release of the enzyme, improved biochemical characteristics (extended pH-activity profile, stability in acidic medium and during storage for 2 years, and it is perspective for further studies. The proposed method for lysozyme immobilization in the carboxymethyl cellulose sodium salt gel allows to obtain a stable, highly efficient product, with high adhesive properties for attachment to the mucous membranes, that is promising for use in biomedicine.

Covalent immobilization of α-amylase on magnetic particles as catalyst for hydrolysis of high-amylose starch.

Science.gov (United States)

Guo, Hui; Tang, Yi; Yu, Yang; Xue, Lu; Qian, Jun-Qing

2016-06-01

Enzyme immobilized on magnetic particles can be used as efficient recoverable biocatalysts under strong magnetic response. To enable re-use of enzyme, modified Fe3O4 particles were used as carrier to immobilize α-amylase in this paper. Firstly, the surface of Fe3O4 particles were coated with amino groups by direct using TEOS (tetraethoxysilane) followed by treatment with APTES (3-aminopropyltriethoxysilane) and then carboxylated by reacting it with succinic anhydride. In addition, the effect of the immobilization condition on enzyme activity recovery and immobilization efficiency were investigated. The results showed that the optimal immobilization occurred under following conditions: pH 5.5, 40°C, enzyme concentration of 20mgmL(-1), reaction time for 36h. Using immobilized α-amylase as biocatalyst, the optimum pH and temperature for hydrolysis were observed to be 6.5 and 60°C. The kinetics of hydrolysis reaction were studied using Michaelis-Menten equation. The affinity constant (Km) and maximum reaction rate (vmax) of magnetic particles immobilization α-amylase (MPIA) was 0.543mgmL(-1) and 1.321mgmin(-1) compared to those of 0.377mgmL(-1) and 6.859mgmin(-1) of free enzyme. After immobilization, enzymatic activity, storage stability, thermo-stability, and reusability of MPIA were found superior to those of the free one. MPIA maintained 86% enzyme activity after 30 days and maintained 78% enzyme activity after recycling six times. Copyright © 2016 Elsevier B.V. All rights reserved.

Immobilization of biomolecules onto surfaces according to ultraviolet light diffraction patterns

International Nuclear Information System (INIS)

Bjoern Petersen, Steffen; Kold di Gennaro, Ane; Neves-Petersen, Maria Teresa; Skovsen, Esben; Parracino, Antonietta

2010-01-01

We developed a method for immobilization of biomolecules onto thiol functionalized surfaces according to UV diffraction patterns. UV light-assisted molecular immobilization proceeds through the formation of free, reactive thiol groups that can bind covalently to thiol reactive surfaces. We demonstrate that, by shaping the pattern of the UV light used to induce molecular immobilization, one can control the pattern of immobilized molecules onto the surface. Using a single-aperture spatial mask, combined with the Fourier transforming property of a focusing lens, we show that submicrometer (0.7 μm) resolved patterns of immobilized prostate-specific antigen biomolecules can be created. If a dual-aperture spatial mask is used, the results differ from the expected Fourier transform pattern of the mask. It appears as a superposition of two diffraction patterns produced by the two apertures, with a fine structured interference pattern superimposed.

Intergenus Protoplast Fusion between Pichia manshurica and Rhodosporidium paludigenum to Increase the Production of Inulinase

Directory of Open Access Journals (Sweden)

Wijanarka Wijanarka

2014-12-01

Full Text Available The purposes of this study was to identify the optimum concentration of the lytic enzyme Glucanex for protoplast isolation and to conduct fusion for the purpose of increasing inulinase production. The study performs the protoplast fusion technique using Pichia manshurica and Rhodosporidium paludigenum. Protoplast fusion consists of a series of stages: protoplast isolation, protoplast fusion, protoplast regeneration, and analysis of hybrid fusion results. Protoplast isolation and fusion success rate are determined by various factors, including age of the culture, media type, and type of lytic enzymes used. Hybrid results were analyzed using a fungicide as a marker and measuring specific growth rate (μ of the hybrid compared with parental growth rates. Results demonstrated that a concentration of 4 mg/mL of Glucanex produces the greatest number of protoplasts, 7.2 x 1010 (cell/mL for P. manshurica and 8.8 x 1010 (cell/mL for Rh. paludigenum. The results of analysis of hybrid fusions indicate that the study has identified a new fusant, called fusant F4. Fusant F4 is capable of producing the highest inulinase, 0.6892 IU, compared with parentals P. manshurica, 0557 IU, and Rh. paludigenum, 0.3263 IU. Fusant F4 has specific growth rate (μ of 0.3360/h and generation time (g of 2.0629 h.

Enhanced starch hydrolysis using α-amylase immobilized on cellulose ultrafiltration affinity membrane.

Science.gov (United States)

Konovalova, Viktoriia; Guzikevich, Kateryna; Burban, Anatoliy; Kujawski, Wojciech; Jarzynka, Karolina; Kujawa, Joanna

2016-11-05

In order to prepare ultrafiltration membranes possessing biocatalytic properties, α-amylase has been immobilized on cellulose membranes. Enzyme immobilization was based on a covalent bonding between chitosan and a surface of cellulose membrane, followed by an attachment of Cibacron Blue F3G-A dye as affinity ligand. Various factors affecting the immobilization process, such as enzyme concentration, pH of modifying solution, zeta-potential of membrane surface, and stability of immobilized enzyme were studied. The applicability of immobilized α-amylase has been investigated in ultrafiltration processes. The immobilization of α-amylase on membrane surface allows to increase the value of mass transfer coefficient and to decrease the concentration polarization effect during ultrafiltration of starch solutions. The enzyme layer on the membrane surface prevents a rapid increase of starch concentration due to the amylase hydrolysis of starch in the boundary layer. The presented affinity immobilization technique allows also for the regeneration of membranes from inactivated enzyme. Copyright © 2016 Elsevier Ltd. All rights reserved.

Porous chitosan beads of superior mechanical properties for the covalent immobilization of enzymes.

Science.gov (United States)

Wahba, Marwa I

2017-12-01

Porous chitosan beads of superior mechanical properties were produced via a two stepped treatment process. First, the chitosan ionotropic gelation solution was supplemented with Na 2 CO 3 , which acted as a porogen. Afterwards, the beads were chemically cross-linked with glutaraldehyde. This treatment also caused the produced porous chitosan beads to acquire higher observed activities of immobilized β-d-galactosidase (β-gal). The observed activities of the β-gal immobilized onto the 0.2M and the 0.35M Na 2 CO 3 treated beads were 1.63 and 1.91 fold respectively, higher than the activity offered by the control beads. Nevertheless, both the control beads and the 0.2M Na 2 CO 3 beads caused the optimum pH range of β-gal to shift from 4.6-5.1 to ∼2.7-5. The enzyme's optimum temperature shifted from 55 to 60°C after its immobilization onto the control chitosan beads whereas the β-gal immobilized onto the 0.2M Na 2 CO 3 chitosan beads exhibited a temperature optimum of 55-60°C. The reusability study revealed the superiority of the 0.2M Na 2 CO 3 treated beads which retained 59.1% of their initial activity during the 13th enzymatic cycle. On the other hand, the control chitosan beads were fragmented and lost their activity after only four enzymatic cycles. Copyright © 2017 Elsevier B.V. All rights reserved.

Consolidated ethanol production from Jerusalem artichoke tubers at elevated temperature by Saccharomyces cerevisiae engineered with inulinase expression through cell surface display.

Science.gov (United States)

Khatun, M Mahfuza; Liu, Chen-Guang; Zhao, Xin-Qing; Yuan, Wen-Jie; Bai, Feng-Wu

2017-02-01

Ethanol fermentation from Jerusalem artichoke tubers was performed at elevated temperatures by the consolidated bioprocessing strategy using Saccharomyces cerevisiae MK01 expressing inulinase through cell surface display. No significant difference was observed in yeast growth when temperature was controlled at 38 and 40 °C, respectively, but inulinase activity with yeast cells was substantially enhanced at 40 °C. As a result, enzymatic hydrolysis of inulin was facilitated and ethanol production was improved with 89.3 g/L ethanol produced within 72 h from 198.2 g/L total inulin sugars consumed. Similar results were also observed in ethanol production from Jerusalem artichoke tubers with 85.2 g/L ethanol produced within 72 h from 185.7 g/L total sugars consumed. On the other hand, capital investment on cooling facilities and energy consumption for running the facilities would be saved, since regular cooling water instead of chill water could be used to cool down the fermentation system.

Immobilization of cellulases on magnetic particles to enable enzyme recycling during hydrolysis of lignocellulose

DEFF Research Database (Denmark)

Alftrén, Johan

feedstocks containing insolubles. This could potentially be overcome by immobilizing the cellulases on magnetically susceptible particles. Consequently, the immobilized cellulases could be magnetically recovered and recycled for a new cycle of enzymatic hydrolysis of cellulose. The main objective...... of this thesis was to examine the possibility of immobilizing cellulases on magnetic particles in order to enable enzyme re-use. Studies at lab and pilot scale (20 L) were conducted using model and real substrates. In paper I and III beta-glucosidase or a whole cellulase mixture was covalently immobilized...... on commercial, but expensive, magnetic particles activated with different chemistries. It was observed that the highest immobilized enzyme activities were obtained using magnetic particles activated with cyanuric chloride. In paper II biotinylated recombinant beta-glucosidase was produced and immobilized...

Micrometer sized immobilization of protein molecules onto quartz, silicium and gold.

Science.gov (United States)

Petersen, Steffen B.; Neves-Petersen, Maria Teresa; Klitgaard, Søren; Duroux, Meg Crookshanks

2006-02-01

We demonstrate that ultraviolet light can be used to make sterically oriented covalent immobilization of a large variety of protein molecules onto either gold or thiolated quartz or silicium. The reaction mechanism behind the reported new technology involves light induced breakage of disulphide bridges in proteins upon UV illumination of nearby aromatic amino acids, resulting in the formation of free, reactive thiol groups that will form covalent bonds with thiol reactive surfaces. The protein molecules in general retain their function. The size of the immobilization spot is determined by the dimension of the UV beam. In principle, the spot size may be as small as 1 micrometer or less. We have developed the necessary technology for preparing large protein arrays of enzymes and fragments of monoclonal antibodies. Dedicated Image Processing Software has been developed for making quality assessment of the protein arrays. A multitude of important application areas such as drug carriers and drug delivery, bioelectronics, carbon nanotubes, nanoparticles as well as protein glue are discussed.

Immobilization of cellulase on functionalized cobalt ferrite nanoparticles

Energy Technology Data Exchange (ETDEWEB)

Bohara, Raghvendra Ashok; Thorat, Nanasaheb Devappa; Pawar, Shivaji Hariba [Center for Interdisciplinary Research, D. Y. Patil University, Kolhapur (India)

2016-01-15

Amine functionalized cobalt ferrite (AF-CoFe{sub 2}O{sub 4}) magnetic nanoparticles (MNPs) were used for immobilization of cellulase enzyme via 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDS) and N-hydroxysuccinimide (NHS) coupling reaction. The structural, morphological and magnetic properties of AF-CoFe{sub 2}O{sub 4} were determined. TEM micrograph revealed a mean diameter of -8 nm and showed that the AF-CoFe{sub 2}O{sub 4} remain distinct with no significant change in size after binding with cellulase. Fourier transform infrared (FT-IR) spectroscopy confirmed the binding of cellulase to AF-CoFe{sub 2}O{sub 4}. The properties of immobilized cellulase were investigated by optimizing binding efficiency, pH, temperature and reusability. The results showed that the immobilized cellulase has higher thermal stability than free cellulase, which might be due to covalent interaction between cellulase and AF-CoFe{sub 2}O{sub 4} surface. The immobilized cellulase also showed good reusability after recovery. Therefore, AF-CoFe{sub 2}O{sub 4} MNPs can be considered as promising candidate for enzyme immobilization.

Improved Procedure for Preparation of Covalently Bonded Cellulose Tris-phenylcarbamate Chiral Stationary Phases

Institute of Scientific and Technical Information of China (English)

秦峰; 陈小明; 刘月启; 邹汉法; 王俊德

2005-01-01

The classical method for preparation of covalently boned cellulose derivative chiral stationary phases (CSP) with diisocyanate as spacer was improved. Diisocyanate was firstly allowed to react with 3-aminopropyltriethoxysilane, and the resulting product was then applied as the spacer reagent to immobilize cellulose derivatives onto silica gel. Influences of the amount and the length of the spacer on the optical resolution ability of the CSP were investigated. Comparing improved procedure to classical diisocyanate method, the cross-linking between the glucose units of the cellulose derivatives was avoided to the most extent. With the improved procedure, regio-nonselective ways could be adooted to prepare covalently bonded CSP, which showed an advantage for the rapid preparation.

Self-assembling peptide hydrogels immobilized on silicon surfaces

International Nuclear Information System (INIS)

Franchi, Stefano; Battocchio, Chiara; Galluzzi, Martina; Navisse, Emanuele; Zamuner, Annj; Dettin, Monica; Iucci, Giovanna

2016-01-01

The hydrogels of self-assembling ionic complementary peptides have collected in the scientific community increasing consensus as mimetics of the extracellular matrix that can offer 3D supports for cell growth or be vehicles for the delivery of stem cells or drugs. Such scaffolds have also been proposed as bone substitutes for small defects as they promote beneficial effects on human osteoblasts. In this context, our research deals with the introduction of a layer of self-assembling peptides on a silicon surface by covalent anchoring and subsequent physisorption. In this work, we present a spectroscopic investigation of the proposed bioactive scaffolds, carried out by surface-sensitive spectroscopic techniques such as XPS (X-ray photoelectron spectroscopy) and RAIRS (Reflection Absorption Infrared Spectroscopy) and by state-of-the-art synchrotron radiation methodologies such as angle dependent NEXAFS (Near Edge X-ray Absorption Fine Structure). XPS studies confirmed the change in the surface composition in agreement with the proposed enrichments, and led to assess the self-assembling peptide chemical stability. NEXAFS spectra, collected in angular dependent mode at the N K-edge, allowed to investigate the self-assembling behavior of the macromolecules, as well as to determine their molecular orientation on the substrate. Furthermore, Infrared Spectroscopy measurements demonstrated that the peptide maintains its secondary structure (β-sheet anti-parallel) after deposition on the silicon surface. The complementary information acquired by means of XPS, NEXAFS and RAIRS lead to hypothesize a “layer-by-layer” arrangement of the immobilized peptides, giving rise to an ordered 3D nanostructure. - Highlights: • A self-assembling peptide (SAP) was covalently immobilized of on a flat silicon surface. • A physisorbed SAP layer was grown on top of the covalently immobilized peptide layer. • Molecular order and orientation of the peptide overlayer on the flat silicon

Self-assembling peptide hydrogels immobilized on silicon surfaces

Energy Technology Data Exchange (ETDEWEB)

Franchi, Stefano; Battocchio, Chiara; Galluzzi, Martina; Navisse, Emanuele [Department of Sciences, University “Roma Tre”, Via della Vasca Navale 79, Roma, 00146 (Italy); Zamuner, Annj; Dettin, Monica [Department of Industrial Engineering, University of Padua, Via Marzolo, 9, Padua, 35131 (Italy); Iucci, Giovanna, E-mail: giovanna.iucci@uniroma3.it [Department of Sciences, University “Roma Tre”, Via della Vasca Navale 79, Roma, 00146 (Italy)

2016-12-01

The hydrogels of self-assembling ionic complementary peptides have collected in the scientific community increasing consensus as mimetics of the extracellular matrix that can offer 3D supports for cell growth or be vehicles for the delivery of stem cells or drugs. Such scaffolds have also been proposed as bone substitutes for small defects as they promote beneficial effects on human osteoblasts. In this context, our research deals with the introduction of a layer of self-assembling peptides on a silicon surface by covalent anchoring and subsequent physisorption. In this work, we present a spectroscopic investigation of the proposed bioactive scaffolds, carried out by surface-sensitive spectroscopic techniques such as XPS (X-ray photoelectron spectroscopy) and RAIRS (Reflection Absorption Infrared Spectroscopy) and by state-of-the-art synchrotron radiation methodologies such as angle dependent NEXAFS (Near Edge X-ray Absorption Fine Structure). XPS studies confirmed the change in the surface composition in agreement with the proposed enrichments, and led to assess the self-assembling peptide chemical stability. NEXAFS spectra, collected in angular dependent mode at the N K-edge, allowed to investigate the self-assembling behavior of the macromolecules, as well as to determine their molecular orientation on the substrate. Furthermore, Infrared Spectroscopy measurements demonstrated that the peptide maintains its secondary structure (β-sheet anti-parallel) after deposition on the silicon surface. The complementary information acquired by means of XPS, NEXAFS and RAIRS lead to hypothesize a “layer-by-layer” arrangement of the immobilized peptides, giving rise to an ordered 3D nanostructure. - Highlights: • A self-assembling peptide (SAP) was covalently immobilized of on a flat silicon surface. • A physisorbed SAP layer was grown on top of the covalently immobilized peptide layer. • Molecular order and orientation of the peptide overlayer on the flat silicon

Photochemical immobilization of anthraquinone conjugated oligonucleotides and PCR amplicons on solid surfaces

DEFF Research Database (Denmark)

Koch, T.; Jacobsen, N.; Fensholdt, J.

2000-01-01

Ligand immobilization on solid surfaces is an essential step in fields such as diagnostics, bio sensor manufacturing, and new material sciences in general. In this paper a photochemical approach based on anthraquinone as the chromophore is presented. Photochemical procedures offer special...... advantages as they are able to generate highly reactive species in an orientation specific manner. As presented here, anthraquinone (AQ) mediated covalent DNA immobilization appears to be superior to currently known procedures. A synthetic procedure providing AQ-phosphoramidites is presented. These reagents...... facilitate AQ conjugation during routine DNA synthesis, thus enabling the AQ-oligonucleotides to be immobilized in a very convenient and efficient manner. AQ-conjugated PCR primers can be used directly in PCR. When the PCR is performed in solution, the amplicons can be immobilized after the PCR. Moreover...

Improvement of the stability and activity of immobilized glucose oxidase on modified iron oxide magnetic nanoparticles

Energy Technology Data Exchange (ETDEWEB)

Abbasi, Mahboube, E-mail: mahbubeabbasi@yahoo.com; Amiri, Razieh, E-mail: razieh.amiri@gmail.com; Bordbar, Abdol-Kalegh, E-mail: bordbar@chem.ui.ac.ir; Ranjbakhsh, Elnaz, E-mail: e.ranjbakhsh@yahoo.com; Khosropour, Ahmad-Reza, E-mail: khosropour@chem.ui.ac.ir

2016-02-28

Graphical abstract: - Highlights: • Modified iron oxide magnetic nanoparticles were synthesized by co-precipitation method and characterized by TEM and XRD. • Covalent attachment of GOX to MIMNs was confirmed by FT-IR technique. • Optimization of the reaction time and initial amount of the GOX were carried out. • Improvement of activity and stability of immobilized GOX have been increased in comparison of free GOX. - Abstract: Immobilized proteins and enzymes are widely investigated in the medical field as well as the food and environmental fields. In this study, glucose oxidase (GOX) was covalently immobilized on the surface of modified iron oxide magnetic nanoparticles (MIMNs) to produce a bioconjugate complex. Transmission electron microscopy (TEM) and X-ray diffraction (XRD) were used to the size, shape and structure characterization of the MIMNs. Binding of GOX to these MIMNs was confirmed by using FT-IR spectroscopy. The stability of the immobilized and free enzyme at different temperature and pH values was investigated by measuring the enzymatic activity. These studies reveal that the enzyme's stability is enhanced by immobilization. Further experiments showed that the storage stability of the enzyme is improved upon binding to the MIMNs. The results of kinetic measurements suggest that the effect of the immobilization process on substrate and product diffusion is small. Such bioconjugates can be considered as a catalytic nanodevice for accelerating the glucose oxidation reaction for biotechnological purposes.

Comparative study on antibody immobilization strategies for efficient circulating tumor cell capture.

Science.gov (United States)

Ates, Hatice Ceren; Ozgur, Ebru; Kulah, Haluk

2018-03-23

Methods for isolation and quantification of circulating tumor cells (CTCs) are attracting more attention every day, as the data for their unprecedented clinical utility continue to grow. However, the challenge is that CTCs are extremely rare (as low as 1 in a billion of blood cells) and a highly sensitive and specific technology is required to isolate CTCs from blood cells. Methods utilizing microfluidic systems for immunoaffinity-based CTC capture are preferred, especially when purity is the prime requirement. However, antibody immobilization strategy significantly affects the efficiency of such systems. In this study, two covalent and two bioaffinity antibody immobilization methods were assessed with respect to their CTC capture efficiency and selectivity, using an anti-epithelial cell adhesion molecule (EpCAM) as the capture antibody. Surface functionalization was realized on plain SiO 2 surfaces, as well as in microfluidic channels. Surfaces functionalized with different antibody immobilization methods are physically and chemically characterized at each step of functionalization. MCF-7 breast cancer and CCRF-CEM acute lymphoblastic leukemia cell lines were used as EpCAM positive and negative cell models, respectively, to assess CTC capture efficiency and selectivity. Comparisons reveal that bioaffinity based antibody immobilization involving streptavidin attachment with glutaraldehyde linker gave the highest cell capture efficiency. On the other hand, a covalent antibody immobilization method involving direct antibody binding by N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC)-N-hydroxysuccinimide (NHS) reaction was found to be more time and cost efficient with a similar cell capture efficiency. All methods provided very high selectivity for CTCs with EpCAM expression. It was also demonstrated that antibody immobilization via EDC-NHS reaction in a microfluidic channel leads to high capture efficiency and selectivity.

In situ one-pot preparation of superparamagnetic hydrophilic porous microspheres for covalently immobilizing penicillin G acylase to synthesize amoxicillin

Science.gov (United States)

Xue, Ping; Gu, Yaohua; Su, Weiguang; Shuai, Huihui; Wang, Julan

2016-01-01

Magnetic hydrophilic porous microspheres were successfully one-pot synthesized for the first time via in situ inverse suspension polymerization of glycidyl methacrylate, N,N‧-methylene bisacrylamide and 2-hydroxyethyl methacrylate in the presence of Fe3+ and Fe2+ dispersed in formamide, which were denoted as magnetic Fe3O4-GMH microspheres. The morphology and properties of magnetic Fe3O4-GMH microspheres were characterized by SEM, VSM, XRD, FTIR, and so on. The formamide content had an important influence on the morphology of Fe3O4-GMH, and nearly perfectly spherical Fe3O4-GMH particles were formed when the amount of formamide was 15 ml. The diameters of the microspheres were in the range of 100-200 μm and Fe3O4-GMH exhibited superparamagnetic behavior with the saturation magnetization of 5.44 emu/g. The specific surface area of microspheres was 138.7 m2/g, the average pore diameter and pore volume were 15.1 nm and 0.60 cm3/g, respectively. The content of oxirane groups on Fe3O4-GMH was 0.40 mmol/g. After penicillin G acylase (PGA) was covalently immobilized on Fe3O4-GMH microspheres, the catalytic performance for amoxicillin synthesis by 6-aminopenicillanic acid and D-hydroxyphenylglycine methyl ester was largely improved. As a result, 90.1% amoxicillin yield and 1.18 of the synthesis/hydrolysis (S/H) ratio were achieved on PGA/Fe3O4-GMH with ethylene glycol as solvent, but only 62.6% amoxicillin yield and 0.37 of the S/H ratio were obtained on free PGA under the same reaction conditions. Furthermore, the amoxicillin yield and S/H ratio were still kept at 88.2% and 1.06, respectively after the immobilized PGA was magnetically separated and recycled for 10 times, indicating that PGA/Fe3O4-GMH had a very good reusability.

Reversible thermal denaturation of immobilized rhodanese

International Nuclear Information System (INIS)

Horowitz, P.; Bowman, S.

1987-01-01

For the first time, the enzyme rhodanese had been refolded after thermal denaturation. This was previously not possible because of the strong tendency for the soluble enzyme to aggregate at temperatures above 37 degrees C. The present work used rhodanese that was covalently coupled to a solid support under conditions that were found to preserve enzyme activity. Rhodanese was immobilized using an N-hydroxymalonimidyl derivative of Sepharose containing a 6-carbon spacer. The number of immobilized competent active sites was measured by using [ 35 S]SO 3 (2-) to form an active site persulfide that is the obligatory catalytic intermediate. Soluble enzyme was irreversibly inactivated in 10 min at 52 degrees C. The immobilized enzyme regained at least 30% of its original activity even after boiling for 20 min. The immobilized enzyme had a Km and Vmax that were each approximately 3 times higher than the corresponding values for the native enzyme. After preincubation at high temperatures, progress curves for the immobilized enzyme showed induction periods of up to 5 min before attaining apparently linear steady states. The pH dependence of the activity was the same for both the soluble and the immobilized enzyme. These results indicate significant stabilization of rhodanese after immobilization, and instabilities caused by adventitious solution components are not the sole reasons for irreversibility of thermal denaturation seen with the soluble enzyme. The results are consistent with models for rhodanese that invoke protein association as a major cause of inactivation of the enzyme. Furthermore, the induction period in the progress curves is consistent with studies which show that rhodanese refolding proceeds through intermediate states

Catalytic properties of immobilized tannase produced from Aspergillus aculeatus compared with the free enzyme

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A. B El-Tanash

2011-09-01

Full Text Available Aspergillus aculeatus tannase was immobilized on several carriers by entrapment and covalent binding with cross - linking. Tannase immobilized on gelatin with cross - linking agent showed the highest activity and immobilization yield. The optimum pH of the immobilized enzyme was shifted to a more acidic range compared with the free enzyme (from pH 5.5 to pH 5.0. The optimum temperature of the reaction was determined to be 50ºC for the free enzyme and 60ºC for the immobilized form. The thermal stability, as well as stability over a wide range of pH, was significantly improved by the immobilization process. The calculated Km of the immobilized tannase (11.8 mg ml-1 is higher than that of the free tannase (6.5 mg ml-1, while Vmax of the immobilized enzyme (0.32 U (µg protein-1 is lower than that of the free tannase (2.7 U (µg protein-1. The immobilized enzyme was able to retain 84 % of the initial catalytic activity after 5.0 cycles.

Covalent Binding of Antibodies to Cellulose Paper Discs and Their Applications in Naked-eye Colorimetric Immunoassays.

Science.gov (United States)

Peng, Yanfen; Gelder, Victor Van; Amaladoss, Anburaj; Patel, Kadamb Haribhai

2016-10-21

This report presents two methods for the covalent immobilization of capture antibodies on cellulose filter paper grade No. 1 (medium-flow filter paper) discs and grade No. 113 (fast-flow filter paper) discs. These cellulose paper discs were grafted with amine functional groups through a silane coupling technique before the antibodies were immobilized on them. Periodate oxidation and glutaraldehyde cross-linking methods were used to graft capture antibodies on the cellulose paper discs. In order to ensure the maximum binding capacity of the capture antibodies to their targets after immobilization, the effects of various concentrations of sodium periodate, glutaraldehyde, and capture antibodies on the surface of the paper discs were investigated. The antibodies that were coated on the amine-functionalized cellulose paper discs through a glutaraldehyde cross-linking agent showed enhanced binding activity to the target when compared to the periodate oxidation method. IgG (in mouse reference serum) was used as a reference target in this study to test the application of covalently immobilized antibodies through glutaraldehyde. A new paper-based, enzyme-linked immunosorbent assay (ELISA) was successfully developed and validated for the detection of IgG. This method does not require equipment, and it can detect 100 ng/ml of IgG. The fast-flow filter paper was more sensitive than the medium-flow filter paper. The incubation period of this assay was short and required small sample volumes. This naked-eye, colorimetric immunoassay can be extended to detect other targets that are identified with conventional ELISA.

High production of D-tagatose, a potential sugar substitute, using immobilized L-arabinose isomerase.

Science.gov (United States)

Kim, P; Yoon, S H; Roh, H J; Choi, J H

2001-01-01

An L-arabinose isomerase of Escherichia coli was immobilized using covalent binding to agarose to produce D-tagatose, a bulking sweetener that can be economically used as a sugar substitute. The immobilized L-arabinose isomerase stably produced an average of 7.5 g-tagatose/L.day for 7 days with a productivity exceeding that of the free enzyme (0.47 vs 0.30 mg/U.day). Using a scaled-up immobilized enzyme system, 99.9 g-tagatose/L was produced from galactose with 20% equilibrium in 48 h. The process was repeated two more times with production of 104.1 and 103.5 g-tagatose/L. D-Tagatose production using an immobilized L-arabinose isomerase has a high potential for commercial application.

Gold nanoparticles covalently assembled onto vesicle structures as possible biosensing platform

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M. Fátima Barroso

2016-05-01

Full Text Available In this contribution a strategy is shown to covalently immobilize gold nanoparticles (AuNPs onto vesicle bilayers with the aim of using this nanomaterial as platform for the future design of immunosensors. A novel methodology for the self-assembly of AuNPs onto large unilamellar vesicle structures is described. The vesicles were formed with 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC and 1-undecanethiol (SH. After, the AuNPs photochemically synthesized in pure glycerol were mixed and anchored onto SH–DOPC vesicles. The data provided by voltammetry, spectrometry and microscopy techniques indicated that the AuNPs were successfully covalently anchored onto the vesicle bilayer and decorated vesicles exhibit a spherical shape with a size of 190 ± 10 nm. The developed procedure is easy, rapid and reproducible to start designing a possible immunosensor by using environmentally friendly procedures.

Application of immobilized synthetic anti-lipopolysaccharide peptides for the isolation and detection of bacteria.

Science.gov (United States)

Sandetskaya, N; Engelmann, B; Brandenburg, K; Kuhlmeier, D

2015-08-01

The molecular detection of microorganisms in liquid samples generally requires their enrichment or isolation. The aim of our study was to evaluate the capture and pre-concentration of bacteria by immobilized particular cationic antimicrobial peptides, called synthetic anti-lipopolysaccharide peptides (SALP). For the proof-of-concept and screening of different SALP, the peptides were covalently immobilized on glass slides, and the binding of bacteria was confirmed by microscopic examination of the slides or their scanning, in case of fluorescent bacterial cells. The most efficient SALP was further tethered to magnetic beads. SALP beads were used for the magnetic capture of Escherichia coli in liquid samples. The efficiency of this strategy was evaluated using polymerase chain reaction (PCR). Covalently immobilized SALP were capable of capturing bacteria in liquid samples. However, PCR was hampered by the unspecific binding of DNA to the positively charged peptide. We developed a method for DNA recovery by the enzymatic digestion of the peptide, which allowed for a successful PCR, though the method had its own adverse impact on the detection and, thus, did not allow for the reliable quantitative analysis of the pathogen enrichment. Immobilized SALP can be used as capture molecules for bacteria in liquid samples and can be recommended for the design of the assays or decontamination of the fluids. For the accurate subsequent detection of bacteria, DNA-independent methods should be used.

Covalent Anchoring of Chloroperoxidase and Glucose Oxidase on the Mesoporous Molecular Sieve SBA-15

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Martin Hartmann

2010-02-01

Full Text Available Functionalization of porous solids plays an important role in many areas, including heterogeneous catalysis and enzyme immobilization. In this study, large-pore ordered mesoporous SBA-15 molecular sieves were synthesized with tetraethyl orthosilicate (TEOS in the presence of the non-ionic triblock co-polymer Pluronic P123 under acidic conditions. These materials were grafted with 3 aminopropyltrimethoxysilane (ATS, 3-glycidoxypropyltrimethoxysilane (GTS and with 3 aminopropyltrimethoxysilane and glutaraldehyde (GA-ATS in order to provide covalent anchoring points for enzymes. The samples were characterized by nitrogen adsorption, powder X-ray diffraction, solid-state NMR spectroscopy, elemental analysis, diffuse reflectance fourier transform infrared spectroscopy and diffuse reflectance UV/Vis spectroscopy. The obtained grafted materials were then used for the immobilization of chloroperoxidase (CPO and glucose oxidase (GOx and the resulting biocatalysts were tested in the oxidation of indole. It is found that enzymes anchored to the mesoporous host by the organic moieties can be stored for weeks without losing their activity. Furthermore, the covalently linked enzymes are shown to be less prone to leaching than the physically adsorbed enzymes, as tested in a fixed-bed reactor under continuous operation conditions.

Haloalkane hydrolysis with an immobilized haloalkane dehalogenase.

Science.gov (United States)

Dravis, B C; Swanson, P E; Russell, A J

2001-11-20

Haloalkane dehalogenase from Rhodococcus rhodochrous was covalently immobilized onto a polyethyleneimine impregnated gamma-alumina support. The dehalogenating enzyme was found to retain greater than 40% of its original activity after immobilization, displaying an optimal loading (max. activity/supported protein) of 70 to 75 mg/g with an apparent maximum (max. protein/support) of 156 mg/g. The substrate, 1,2,3-trichloropropane, was found to favorably partition (adsorb) onto the inorganic alumina carrier (10 to 20 mg/g), thereby increasing the local reactant concentration with respect to the catalyst's environment, whereas the product, 2,3-dichloropropan-1-ol, demonstrated no affinity. Additionally, the inorganic alumina support exhibited no adverse effects because of solvent/component incompatibilities or deterioration due to pH variance (pH 7.0 to 10.5). As a result of the large surface area to volume ratio of the support matrix and the accessibility of the bound protein, the immobilized biocatalyst was not subject to internal mass transfer limitations. External diffusional restrictions could be eliminated with simple agitation (mixing speed: 50 rpm; flux: 4.22 cm/min). The pH-dependence of the immobilized dehalogenase was essentially the same as that for the native enzyme. Finally, both the thermostability and resistance toward inactivation by organic solvent were improved by more than an order of magnitude after immobilization. Copyright 2001 John Wiley & Sons, Inc.

Immobilization of trypsin on sub-micron skeletal polymer monolith

Energy Technology Data Exchange (ETDEWEB)

Yao Chunhe [Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190 (China); Graduate School, Chinese Academy of Sciences, Beijing 100049 (China); Qi Li, E-mail: qili@iccas.ac.cn [Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190 (China); Hu Wenbin [Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190 (China); Graduate School, Chinese Academy of Sciences, Beijing 100049 (China); Wang Fuyi [Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190 (China); Yang Gengliang [College of Pharmacy, Hebei University, Baoding 071002 (China)

2011-04-29

A new kind of immobilized trypsin reactor based on sub-micron skeletal polymer monolith has been developed. Covalent immobilization of trypsin on this support was performed using the epoxide functional groups in either a one- or a multi-step reaction. The proteolytic activity of the immobilized trypsin was measured by monitoring the formation of N-{alpha}-benzoyl-L-arginine (BA) which is the digestion product of a substrate N-{alpha}-benzoyl-L-arginine ethyl ester (BAEE). Results showed that the digestion speed was about 300 times faster than that performed in free solution. The performance of such an enzyme reactor was further demonstrated by digesting protein myoglobin. It has been found that the protein digestion could be achieved in 88 s at 30 deg. C, which is comparable to 24 h digestion in solution at 37 {sup o}C. Furthermore, the immobilized trypsin exhibits increased stability even after continuous use compared to that in free solution. The present monolithic enzyme-reactor provides a promising platform for the proteomic research.

Highly selective and sensitive optical sensor for determination of Pb2+and Hg2+ ions based on the covalent immobilization of dithizone on agarose membrane

Science.gov (United States)

Zargoosh, Kiomars; Babadi, Fatemeh Farhadian

2015-02-01

A highly sensitive and selective optical membrane for determination of Hg2+ and Pb2+ was prepared by covalent immobilization of dithizone on agarose membrane. In addition to its high stability, reproducibility and relatively long lifetime, the proposed optical sensor revealed good selectivity for target ions over a large number of alkali, alkaline earth, transition, and heavy metal ions. The proposed optical membrane displays linear responses from 1.1 × 10-8 to 2.0 × 10-6 mol L-1 and 1.2 × 10-8 to 2.4 × 10-6 mol L-1 for Hg2+ and Pb2+, respectively. The limits of detection (LOD) were 2.0 × 10-9 mol L-1 and 4.0 × 10-9 mol L-1 for Hg2+ and Pb2, respectively. The prepared optical membrane was successfully applied to the determination of Hg2+ and Pb2+ in industrial wastes, spiked tap water and natural waters without any preconcentration step.

Continuous production of chitooligosaccharides by an immobilized enzyme in a dual-reactor system

DEFF Research Database (Denmark)

Santos-Moriano, Paloma; Woodley, John; Plou, Francisco J.

2016-01-01

A chitosanolytic activity found in a commercial α-amylase from Bacillus amylolyquefaciens (BAN) was covalently immobilized onto glyoxal agarose beads (25% recovery of activity) and assessed for the continuous production of chitooligosaccharides (COS). The immobilization did not change the reactio......, the productivity of the PBR at the lowest dilution rate was 37 gCOS L−1 h−1, with a conversion yield of 73%. In contrast, at the highest dilution rate, the productivity was nearly 200 gCOS L−1 h−1, but the conversion yield dropped to around 40%....

Immobilization of cellulase mixtures on magnetic particles for hydrolysis of lignocellulose and ease of recycling

DEFF Research Database (Denmark)

Alftrén, Johan; Hobley, Timothy John

2014-01-01

In the present study whole cellulase mixtures were covalently immobilized on non-porous magnetic particles to enable enzyme reuse. It was shown that CellicCTec2 immobilized on magnetic particles activated with cyanuric chloride gave the highest bead activity measured by mass of reducing sugar...... serum albumin (BSA)) on hydrolysis yield was studied for free and immobilized CellicCTec2. It was observed that for both free and immobilized CellicCTec2 the hydrolysis yield was increased when Tween 80, PEG 6000 or BSA was included. Interaction between magnetic particles (containing immobilized Cellic......CTec2) and lignin was examined and it was demonstrated that addition of BSA completely inhibited interaction while Tween 80 and PEG 6000 had no effect on decreasing magnetic particle-lignin interaction. Hydrolysis of pretreated wheat straw biomass was performed in two consecutive cycles using...

Catalytic Properties and Immobilization Studies of Catalase from Malva sylvestris L.

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G. Arabaci

2013-01-01

Full Text Available Catalase was partially purified from Malva sylvestris L. and immobilized onto chitosan. Then, its catalytic properties were investigated. (NH42SO4 precipitation and dialysis were performed in the extracted enzyme. Further purification was performed with sephadex G-200 column. Kinetic studies of the purified enzyme activity were measured and characterized. The inhibitory effects of KCN, NaN3, CuSO4, and EDTA on M. sylvestris L. catalase activity were observed except NaCl. Furthermore, M. sylvestris L. catalase was immobilized covalently with glutaraldehyde onto chitosan particles. The pH and temperature optima as well as the changes in the kinetics (Km, Vmax of the immobilized and free M. sylvestris L. catalase were determined. The Km value for immobilized catalase (23.4 mM was higher than that of free enzyme (17.6 mM. Optimum temperature was observed higher than that of the free enzyme. The optimum pH was the same for both free and immobilized catalases (pH 7.50. Immobilized catalase showed higher storage and thermal stabilities than free catalases. Free catalase lost all its activity within 60 days whereas immobilized catalase lost 45% of its activity during the same incubation period at 4°C. The remaining immobilized catalase activity was about 70% after 8 cycles of batch operations.

Impedimetric Urea Biosensor Based on Modified Gold Electrode with Urease Immobilized on Glutathione Layer

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Houcine BARHOUMI

2014-05-01

Full Text Available In this work, a glutathione (GSH modified gold microelectrode was used for the covalent immobilization of urease biomolecules via the glutaraldehyde-coupling agent. The self- assembled monolayers (SAMs onto the gold surface was investigated by using the electrochemical impedance spectroscopy measurements (EIS. Before urease grafting, a significant interaction was noticed between urea and the glutathione layer by forming hydrogen bonds. The H-NMR analysis was carried out to highlight the possibility of having a covalent link between urea and the GSH deposited layer. In addition, contact angle measurements were carried out to determine the hydrophobic/hydrophilic feature of the modified gold surface electrode. After urease immobilization a stable and high sensitive impedimetric urea biosensors was obtained with a sensitivity of 8.73´10- 8 W-1mM-1 for the low concentrations range and a sensitivity of 7.03´10-9 W-1mM-1 for the high concentrations range.

Improved Stabilities of Immobilized Glucoamylase on Functionalized Mesoporous Silica Synthesised using Decane as Swelling Agent

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Reni George

2013-06-01

Full Text Available Ordered mesoporous silica, with high porosity was used to immobilize glucoamylase via adsorption and covalent binding. Immobilization of glucoamylase within mesoporous silica was successfully achieved, resulting in catalytically high efficiency during starch hydrolysis. In this study, mesoporous silica was functionalized by co-condensation of tetraethoxysilane (TEOS with organosilane (3-aminopropyl triethoxysilane (APTES in a wide range of molar ratios of APTES: TEOS in the presence of triblock copolymer P123 under acidic hydrothermal conditions. The prepared materials were characterized by Small angle XRD, Nitrogen adsorption – desorption and 29Si MAS solid state NMR. N2 desorption studies showed that pore size distribution decreases due to pore blockage after functionalization and enzyme immobilization. Small angle XRD and 29Si MAS NMR study reveals mesophase formation and Si environment of the materials. The main aim of our work was to study the catalytical activity, effect of pH, temperature storage stability and reusability of covalently bound glucoamylase on mesoporous silica support. The result shows that the stability of enzyme can be enhanced by immobilization.  © 2013 BCREC UNDIP. All rights reservedReceived: 3rd December 2012; Revised: 4th April 2013; Accepted: 20th April 2013[How to Cite: George, R., Gopinath, S., Sugunan, S. (2013. Improved Stabilities of Immobilized Glucoamyl-ase on Functionalized Mesoporous Silica Synthesized using Decane as Swelling Agent. Bulletin of Chemical Reaction Engineering & Catalysis, 8 (1: 70-76. (doi:10.9767/bcrec.8.1.4208.70-76][Permalink/DOI: http://dx.doi.org/10.9767/bcrec.8.1.4208.70-76] | View in  |

Linker-free covalent immobilization of heparin, SDF-1α, and CD47 on PTFE surface for antithrombogenicity, endothelialization and anti-inflammation.

Science.gov (United States)

Gao, Ang; Hang, Ruiqiang; Li, Wan; Zhang, Wei; Li, Penghui; Wang, Guomin; Bai, Long; Yu, Xue-Feng; Wang, Huaiyu; Tong, Liping; Chu, Paul K

2017-09-01

Small-diameter vascular grafts made of biomedical polytetrafluoroethylene (PTFE) suffer from the poor long-term patency rate originating from thrombosis and intimal hyperplasia, which can be ascribed to the insufficient endothelialization and chronic inflammation of the materials. Hence, bio-functionalization of PTFE grafts is highly desirable to circumvent these disadvantages. In this study, a versatile "implantation-incubation" approach in which the biomedical PTFE is initially modified by plasma immersion ion implantation (PIII) is described. After the N 2 PIII treatment, the surface of biomedical PTFE is roughened with nanostructures and more importantly, the abundant free radicals generated underneath the surface continuously migrate to the surface and react with environmental molecules. Taking advantage of this mechanism, various biomolecules with different functions can be steadily immobilized on the surface of PTFE by simple solution immersion. As examples, three typical biomolecules, heparin, SDF-1α, and CD47, are covalently grafted onto the PTFE. In addition to retaining the bioactivity, the surface-functionalized PTFE exhibits reduced thrombogenicity, facilitates the recruitment of endothelial progenitor cells, and even alleviates the inflammatory immune responses of monocytes-macrophages and is thus promising to the development of small-diameter prosthetic vascular grafts with good long-term patency. Copyright © 2017 Elsevier Ltd. All rights reserved.

Development of electrochemical immunosensors based on different serum antibody immobilization methods for detection of Japanese encephalitis virus

International Nuclear Information System (INIS)

Tran, Quang Huy; Hanh Nguyen, Thi Hong; Phan, Thi Nga; Mai, Anh Tuan; Nguyen, Thi Thuy; Vu, Quang Khue

2012-01-01

This paper describes the development of electrochemical immunosensors based on human serum antibodies with different immobilization methods for detection of Japanese encephalitis virus (JEV). Human serum containing anti-JEV antibodies was used to immobilize onto the surface of silanized interdigitated electrodes by four methods: direct adsorption (APTES-serum), covalent binding with a cross linker of glutaraldehyde (APTES-GA-serum), covalent binding with a cross linker of glutaraldehyde combined with anti-human IgG (APTES-GA-anti-HIgG-serum) and covalent binding with a cross linker of glutaraldehyde combined with a bioaffinity of protein A (APTES-GA-PrA-serum). Atomic force microscopy was used to verify surface characteristics of the interdigitated electrodes before and after treatment with serum antibodies. The output signal of the immunosensors was measured by the change of conductivity resulting from the specific binding of JEV antigens and serum antibodies immobilized on the electrodes, with the help of horseradish peroxidase (HRP)-labeled secondary antibody against JEV. The results showed that the APTES-GA-PrA-serum method provided the highest signal of the electrochemical immunosensor for detection of JEV antigens, with the linear range from 25 ng ml −1 to 1 μg ml −1 , and the limit of detection was about 10 ng ml −1 . This study shows a potential development of novel electrochemical immunosensors applied for virus detection in clinical samples in case of possible outbreaks

Characterization of immobilization methods of antiviral antibodies in serum for electrochemical biosensors

Energy Technology Data Exchange (ETDEWEB)

Huy, Tran Quang, E-mail: huytq@nihe.org.vn [National Institute of Hygiene and Epidemiology (NIHE), No1 Yersin St., Hanoi (Viet Nam); International Training Institute for Materials Science (ITIMS), Hanoi University of Science and Technology (HUST), No1 Dai Co Viet, Hanoi (Viet Nam); Hanh, Nguyen Thi Hong; Van Chung, Pham; Anh, Dang Duc; Nga, Phan Thi [National Institute of Hygiene and Epidemiology (NIHE), No1 Yersin St., Hanoi (Viet Nam); Tuan, Mai Anh, E-mail: tuanma-itims@mail.hut.edu.vn [International Training Institute for Materials Science (ITIMS), Hanoi University of Science and Technology (HUST), No1 Dai Co Viet, Hanoi (Viet Nam)

2011-06-01

In this paper, we describes different methods to immobilize Japanese encephalitis virus (JEV) antibodies in human serum onto the interdigitated surface of a microelectrode sensor for optimizing electrochemical detection: (1) direct covalent binding to the silanized surface, (2) binding to the silanized surface via a cross-linker of glutaraldehyde (GA), (3) binding to glutaraldehyde/silanized surface via goat anti-human IgG polyclonal antibody and (4) binding to glutaraldehyde/silanized surface via protein A (PrA). Field emission scanning electron microscopy, Fourier transform infrared spectrometry, and fluorescence microscopy are used to verify the characteristics of antibodies on the interdigitated surface after the serum antibodies immobilization. The analyzed results indicate that the use of protein A is an effective choice for immobilization and orientation of antibodies in serum for electrochemical biosensors. This study provides an advantageous immobilization method of serum containing antiviral antibodies to develop electrochemical biosensors for preliminary screening of viruses in clinical samples from outbreaks.

Silica Nanofibers with Immobilized Tetracycline for Wound Dressing

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Irena Lovětinská-Šlamborová

2016-01-01

Full Text Available Local antibiotic treatment has its justification for superficial infections. The advantage of this treatment is that the antibiotic has effects on bacterial agent directly at the application site. Skin infections which are intended for the local antibiotic treatment are superficial pyoderma, some festering wounds, burns of second and third degree, infected leg ulcers, or decubitus of second and third degree. Tetracyclines are available topical antibiotics with a broad bacterial spectrum. At present, ointments containing tetracycline are also used for the treatment, which rarely can lead to skin sensitization. In this paper, a development of novel nanofibrous material with immobilized tetracycline is presented. Two different methods of immobilized tetracycline quantification onto silica nanofibers are employed. It was proven that the prevailing part of tetracycline was bound weakly by physisorption forces, while the minor part was covalently bound by NH2 groups formed by the preceding functionalization. The silica nanofibers with immobilized tetracycline are promising material for wound dressing applications due to its antibacterial activity; it was proved by tests.

An orientation analysis method for protein immobilized on quantum dot particles

Energy Technology Data Exchange (ETDEWEB)

Aoyagi, Satoka, E-mail: aoyagi@life.shimane-u.ac.jp [Faculty of Life and Environmental Science, Shimane University, 1060 Matsue-shi, Shimane 690-8504 (Japan); Inoue, Masae [Toyota Central R and D Labs., Inc., Nagakute, Aichi 480-1192 (Japan)

2009-11-30

The evaluation of orientation of biomolecules immobilized on nanodevices is crucial for the development of high performance devices. Such analysis requires ultra high sensitivity so as to be able to detect less than one molecular layer on a device. Time-of-flight secondary ion mass spectrometry (TOF-SIMS) has sufficient sensitivity to evaluate the uppermost surface structure of a single molecular layer. The objective of this study is to develop an orientation analysis method for proteins immobilized on nanomaterials such as quantum dot particles, and to evaluate the orientation of streptavidin immobilized on quantum dot particles by means of TOF-SIMS. In order to detect fragment ions specific to the protein surface, a monoatomic primary ion source (Ga{sup +}) and a cluster ion source (Au{sub 3}{sup +}) were employed. Streptavidin-immobilized quantum dot particles were immobilized on aminosilanized ITO glass plates at amino groups by covalent bonding. The reference samples streptavidin directly immobilized on ITO plates were also prepared. All samples were dried with a freeze dryer before TOF-SIMS measurement. The positive secondary ion spectra of each sample were obtained using TOF-SIMS with Ga{sup +} and Au{sub 3}{sup +}, respectively, and then they were compared so as to characterize each sample and detect the surface structure of the streptavidin immobilized with the biotin-immobilized quantum dots. The chemical structures of the upper surface of the streptavidin molecules immobilized on the quantum dot particles were evaluated with TOF-SIMS spectra analysis. The indicated surface side of the streptavidin molecules immobilized on the quantum dots includes the biotin binding site.

A preliminary study of continuous milk coagulation using Cynara cardunculus flower extract and calf rennet immobilized on magnetic particles.

Science.gov (United States)

Liburdi, Katia; Emiliani Spinelli, Sara; Benucci, Ilaria; Lombardelli, Claudio; Esti, Marco

2018-01-15

The aim of this study was to develop a bioreactor design for continuous milk coagulation using a biocatalyst composed of immobilized animal and vegetable rennet on aminated magnetic particles, which has been proven to be an appropriate carrier for enzyme immobilization. Calf and vegetable (Cynara cardunculus) rennets were covalently immobilized on CLEA® magnetic supports and the immobilization procedure was optimized in batch mode, by evaluating protein loading, caseinolytic activity and the coagulation properties of skim milk powder and cow's milk. Subsequently the optimal temperature of immobilized coagulant was defined and a technically-friendly enzyme bioreactor was developed in order to carry out a continuous milk coagulation process with the aim of producing soft cheese. Copyright © 2017 Elsevier Ltd. All rights reserved.

Development and characterization of methacrylate-based hydrazide monoliths for oriented immobilization of antibodies.

Science.gov (United States)

Brne, P; Lim, Y-P; Podgornik, A; Barut, M; Pihlar, B; Strancar, A

2009-03-27

Convective interaction media (CIM; BIA Separations) monoliths are attractive stationary phases for use in affinity chromatography because they enable fast affinity binding, which is a consequence of convectively enhanced mass transport. This work focuses on the development of novel CIM hydrazide (HZ) monoliths for the oriented immobilization of antibodies. Adipic acid dihydrazide (AADH) was covalently bound to CIM epoxy monoliths to gain hydrazide groups on the monolith surface. Two different antibodies were afterwards immobilized to hydrazide functionalized monolithic columns and prepared columns were tested for their selectivity. One column was further tested for the dynamic binding capacity.

Poly(acrylonitrile)chitosan composite membranes for urease immobilization.

Science.gov (United States)

Gabrovska, Katya; Georgieva, Aneliya; Godjevargova, Tzonka; Stoilova, Olya; Manolova, Nevena

2007-05-10

(Poly)acrylonitrile/chitosan (PANCHI) composite membranes were prepared. The chitosan layer was deposited on the surface as well as on the pore walls of the base membrane. This resulted in the reduction of the pore size of the membrane and in an increase of their hydrophilicity. The pore structure of PAN and PANCHI membranes were determined by TEM and SEM analyses. It was found that the average size of the pore under a selective layer base PAN membrane is 7 microm, while the membrane coated with 0.25% chitosan shows a reduced pore size--small or equal to 5 microm and with 0.35% chitosan--about 4 microm. The amounts of the functional groups, the degree of hydrophilicity and transport characteristics of PAN/Chitosan composite membranes were determined. Urease was covalently immobilized onto all kinds of PAN/chitosan composite membranes using glutaraldehyde. Both the amount of bound protein and relative activity of immobilized urease were measured. The highest activity (94%) was measured for urease bound to PANCHI2 membranes (0.25% chitosan). The basic characteristics (pH(opt), pH(stability), T(opt), T(stability), heat inactivation and storage stability) of immobilized urease were determined. The obtained results show that the poly(acrylonitrile)chitosan composite membranes are suitable for enzyme immobilization.

End-Point Immobilization of Recombinant Thrombomodulin via Sortase-Mediated Ligation

Science.gov (United States)

Jiang, Rui; Weingart, Jacob; Zhang, Hailong; Ma, Yong; Sun, Xue-Long

2012-01-01

We report an enzymatic end-point modification and immobilization of recombinant human thrombomodulin (TM), a cofactor for activation of anticoagulant protein C pathway via thrombin. First, a truncated TM mutant consisting of epidermal growth factor-like domains 4–6 (TM456) with a conserved pentapeptide LPETG motif at its C-terminal was expressed and purified in E. coli. Next, the truncated TM456 derivative was site-specifically modified with N-terminal diglycine containing molecules such as biotin and the fluorescent probe dansyl via sortase A (SrtA) mediated ligation (SML). The successful ligations were confirmed by SDS-PAGE and fluorescence imaging. Finally, the truncated TM456 was immobilized onto N-terminal diglycine-functionalized glass slide surface via SML directly. Alternatively, the truncated TM456 was biotinylated via SML and then immobilized onto streptavidin-functionalized glass slide surface indirectly. The successful immobilizations were confirmed by fluorescence imaging. The bioactivity of the immobilized truncated TM456 was further confirmed by protein C activation assay, in which enhanced activation of protein C by immobilized recombinant TM was observed. The sortase A-catalyzed surface ligation took place under mild conditions and is rapid occurring in a single step without prior chemical modification of the target protein. This site-specific covalent modification leads to molecules being arranged in a definitively ordered fashion and facilitating the preservation of the protein’s biological activity. PMID:22372933

Solid-phase synthesis of protein-polymers on reversible immobilization supports.

Science.gov (United States)

Murata, Hironobu; Carmali, Sheiliza; Baker, Stefanie L; Matyjaszewski, Krzysztof; Russell, Alan J

2018-02-27

Facile automated biomacromolecule synthesis is at the heart of blending synthetic and biologic worlds. Full access to abiotic/biotic synthetic diversity first occurred when chemistry was developed to grow nucleic acids and peptides from reversibly immobilized precursors. Protein-polymer conjugates, however, have always been synthesized in solution in multi-step, multi-day processes that couple innovative chemistry with challenging purification. Here we report the generation of protein-polymer hybrids synthesized by protein-ATRP on reversible immobilization supports (PARIS). We utilized modified agarose beads to covalently and reversibly couple to proteins in amino-specific reactions. We then modified reversibly immobilized proteins with protein-reactive ATRP initiators and, after ATRP, we released and analyzed the protein polymers. The activity and stability of PARIS-synthesized and solution-synthesized conjugates demonstrated that PARIS was an effective, rapid, and simple method to generate protein-polymer conjugates. Automation of PARIS significantly reduced synthesis/purification timelines, thereby opening a path to changing how to generate protein-polymer conjugates.

Gelatin Functionalization of Biomaterial Surfaces: Strategies for Immobilization and Visualization

Directory of Open Access Journals (Sweden)

Peter Dubruel

2011-01-01

Full Text Available In the present work, the immobilization of gelatin as biopolymer on two types of implantable biomaterials, polyimide and titanium, was compared. Both materials are known for their biocompatibility while lacking cell-interactive behavior. For both materials, a pre-functionalization step was required to enable gelatin immobilization. For the polyimide foils, a reactive succinimidyl ester was introduced first on the surface, followed by covalent grafting of gelatin. For the titanium material, methacrylate groups were first introduced on the Ti surface through a silanization reaction. The applied functionalities enabled the subsequent immobilization of methacrylamide modified gelatin. Both surface modified materials were characterized in depth using atomic force microscopy, static contact angle measurements, confocal fluorescence microscopy, attenuated total reflection infrared spectroscopy and X-ray photo-electron spectroscopy. The results indicated that the strategies elaborated for both material classes are suitable to apply stable gelatin coatings. Interestingly, depending on the material class studied, not all surface analysis techniques are applicable.

Triclosan-immobilized polyamide thin film composite membranes with enhanced biofouling resistance

Science.gov (United States)

Park, Sang-Hee; Hwang, Seon Oh; Kim, Taek-Seung; Cho, Arah; Kwon, Soon Jin; Kim, Kyoung Taek; Park, Hee-Deung; Lee, Jung-Hyun

2018-06-01

We report on a strategy to improve biofouling resistance of a polyamide (PA) thin-film composite (TFC) reverse osmosis (RO) membrane via chemically immobilizing triclosan (TC), known as a common organic biocide, on its surface. To facilitate covalent attachment of TC on the membrane surface, TC was functionalized with amine moiety to prepare aminopropyl TC. Then, the TC-immobilized TFC (TFC-TC) membranes were fabricated through a one-step amide formation reaction between amine groups of aminopropyl TC and acyl chloride groups present on the PA membrane surface, which was confirmed by high-resolution XPS. Strong stability of the immobilized TC was also confirmed by a hydraulic washing test. Although the TFC-TC membrane showed slightly reduced separation performance compared to the pristine control, it still maintained a satisfactory RO performance level. Importantly, the TFC-TC membrane exhibited excellent antibacterial activity against both gram negative (E. coli and P. aeruginosa) and gram positive (S. aureus) bacteria along with greatly enhanced resistance to biofilm formation. Our immobilization approach offers a robust and relatively benign strategy to control biofouling of functional surfaces, films and membranes.

Effects of nitrogen and carbon sources on the production of inulinase from strain Bacillus sp. SG113

Science.gov (United States)

Gavrailov, Simeon; Ivanova, Viara

2016-03-01

The effects of the carbon and nitrogen substrates on the growth of Bacillus sp. SG113 strain were studied. The use of organic nitrogen sources (peptone, beef extract, yeast extract, casein) leads to rapid cellular growth and the best results for the Bacillus strain were obtained with casein hydrolysate. From the inorganic nitrogen sources studied, the (NH4) 2SO4 proved to be the best nitrogen source. Casein hydrolysate and (NH4) 2SO4 stimulated the invertase synthesis. In the presence of Jerusalem artichoke, onion and garlic extracts as carbon sources the strain synthesized from 6 to 10 times more inulinase.

Calix[6]arene mono-diazonium salt synthesis and covalent immobilization onto glassy carbon electrodes

International Nuclear Information System (INIS)

Cannizzo, Caroline; Jasmin, Jean-Philippe; Vautrin-Ul, Christine; Chausse, Annie; Wagner, Mathieu; Doizi, Denis; Lamouroux, Christine

2014-01-01

This Letter describes the fast synthesis of a mono-aminated calix[6]arene. The immobilization of this macrocycle onto glassy carbon electrodes via diazonium salt chemistry and the electrochemical characterization of the grafted organic layer are also reported. (authors)

Enhancing the functional properties of thermophilic enzymes by chemical modification and immobilization.

Science.gov (United States)

Cowan, Don A; Fernandez-Lafuente, Roberto

2011-09-10

The immobilization of proteins (mostly typically enzymes) onto solid supports is mature technology and has been used successfully to enhance biocatalytic processes in a wide range of industrial applications. However, continued developments in immobilization technology have led to more sophisticated and specialized applications of the process. A combination of targeted chemistries, for both the support and the protein, sometimes in combination with additional chemical and/or genetic engineering, has led to the development of methods for the modification of protein functional properties, for enhancing protein stability and for the recovery of specific proteins from complex mixtures. In particular, the development of effective methods for immobilizing large multi-subunit proteins with multiple covalent linkages (multi-point immobilization) has been effective in stabilizing proteins where subunit dissociation is the initial step in enzyme inactivation. In some instances, multiple benefits are achievable in a single process. Here we comprehensively review the literature pertaining to immobilization and chemical modification of different enzyme classes from thermophiles, with emphasis on the chemistries involved and their implications for modification of the enzyme functional properties. We also highlight the potential for synergies in the combined use of immobilization and other chemical modifications. Copyright © 2011 Elsevier Inc. All rights reserved.

CovalentDock Cloud: a web server for automated covalent docking.

Science.gov (United States)

Ouyang, Xuchang; Zhou, Shuo; Ge, Zemei; Li, Runtao; Kwoh, Chee Keong

2013-07-01

Covalent binding is an important mechanism for many drugs to gain its function. We developed a computational algorithm to model this chemical event and extended it to a web server, the CovalentDock Cloud, to make it accessible directly online without any local installation and configuration. It provides a simple yet user-friendly web interface to perform covalent docking experiments and analysis online. The web server accepts the structures of both the ligand and the receptor uploaded by the user or retrieved from online databases with valid access id. It identifies the potential covalent binding patterns, carries out the covalent docking experiments and provides visualization of the result for user analysis. This web server is free and open to all users at http://docking.sce.ntu.edu.sg/.

Thermostable trypsin conjugates immobilized to biogenic magnetite show a high operational stability and remarkable reusability for protein digestion

Science.gov (United States)

Pečová, M.; Šebela, M.; Marková, Z.; Poláková, K.; Čuda, J.; Šafářová, K.; Zbořil, R.

2013-03-01

In this work, magnetosomes produced by microorganisms were chosen as a suitable magnetic carrier for covalent immobilization of thermostable trypsin conjugates with an expected applicability for efficient and rapid digestion of proteins at elevated temperatures. First, a biogenic magnetite was isolated from Magnetospirillum gryphiswaldense and its free surface was coated with the natural polysaccharide chitosan containing free amino and hydroxy groups. Prior to covalent immobilization, bovine trypsin was modified by conjugating with α-, β- and γ-cyclodextrin. Modified trypsin was bound to the magnetic carriers via amino groups using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysulfosuccinimide as coupling reagents. The magnetic biomaterial was characterized by magnetometric analysis and electron microscopy. With regard to their biochemical properties, the immobilized trypsin conjugates showed an increased resistance to elevated temperatures, eliminated autolysis, had an unchanged pH optimum and a significant storage stability and reusability. Considering these parameters, the presented enzymatic system exhibits properties that are superior to those of trypsin forms obtained by other frequently used approaches. The proteolytic performance was demonstrated during in-solution digestion of model proteins (horseradish peroxidase, bovine serum albumin and hen egg white lysozyme) followed by mass spectrometry. It is shown that both magnetic immobilization and chemical modification enhance the characteristics of trypsin making it a promising tool for protein digestion.

Construction of an Immobilized Thermophilic Esterase on Epoxy Support for Poly(ε-caprolactone Synthesis

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Hui Ren

2016-06-01

Full Text Available Developing an efficient immobilized enzyme is of great significance for improving the operational stability of enzymes in poly(ε-caprolactone synthesis. In this paper, a thermophilic esterase AFEST from the archaeon Archaeoglobus fulgidus was successfully immobilized on the epoxy support Sepabeads EC-EP via covalent attachment, and the immobilized enzyme was then employed as a biocatalyst for poly(ε-caprolactone synthesis. The enzyme loading and recovered activity of immobilized enzyme was measured to be 72 mg/g and 10.4 U/mg using p-nitrophenyl caprylate as the substrate at 80 °C, respectively. Through the optimization of reaction conditions (enzyme concentration, temperature, reaction time and medium, poly(ε-caprolactone was obtained with 100% monomer conversion and low number-average molecular weight (Mn < 1300 g/mol. Further, the immobilized enzyme exhibited excellent reusability, with monomer conversion values exceeding 75% during 15 batch reactions. Finally, poly(ε-caprolactone was enzymatically synthesized with an isolated yield of 75% and Mn value of 3005 g/mol in a gram-scale reaction.

Photochemical immobilization of bovine serum albumin on Ti-O and evaluations in vitro and in vivo

International Nuclear Information System (INIS)

Weng, Y.J.; Qi, F.; Huang, N.; Wang, J.; Cheng, J.Y.; Leng, Y.X.

2008-01-01

Antithrombogenic biomaterials have been of great interest in the development of artificial organs and devices. In this study, titanium oxide coatings were used as the basis for covalent immobilization of a BSA layer by a photochemical method. BSA was first modified with azidophenyl group on the side chain, so it (AZ-BSA) has the property of photo-reactivity. Simultaneously, an organic monolayer of 3-aminopropylphosphonic acid (APP) was introduced on the Ti-O film by self-assembling, and then UV irradiation was used to couple AZ-BSA with APP. FTIR, XPS and contact angle measurements confirmed the occurrence of the modification. Additionally, a surface with both BSA immobilized and non-BSA immobilized regions was prepared by using a mask when irradiating, thus the interactions of materials and platelets were visualized. Platelet experiments of both qualitative and quantitative analysis showed that BSA immobilized surface was effective to inhibit platelet adhesion in vitro. In vivo study also confirmed better hemocompatibility of BSA immobilized surface after 90 days implantation

Improvement of the stability and activity of immobilized glucose oxidase on modified iron oxide magnetic nanoparticles

Science.gov (United States)

Abbasi, Mahboube; Amiri, Razieh; Bordbar, Abdol-Kalegh; Ranjbakhsh, Elnaz; Khosropour, Ahmad-Reza

2016-02-01

Immobilized proteins and enzymes are widely investigated in the medical field as well as the food and environmental fields. In this study, glucose oxidase (GOX) was covalently immobilized on the surface of modified iron oxide magnetic nanoparticles (MIMNs) to produce a bioconjugate complex. Transmission electron microscopy (TEM) and X-ray diffraction (XRD) were used to the size, shape and structure characterization of the MIMNs. Binding of GOX to these MIMNs was confirmed by using FT-IR spectroscopy. The stability of the immobilized and free enzyme at different temperature and pH values was investigated by measuring the enzymatic activity. These studies reveal that the enzyme's stability is enhanced by immobilization. Further experiments showed that the storage stability of the enzyme is improved upon binding to the MIMNs. The results of kinetic measurements suggest that the effect of the immobilization process on substrate and product diffusion is small. Such bioconjugates can be considered as a catalytic nanodevice for accelerating the glucose oxidation reaction for biotechnological purposes.

pH-dependent immobilization of urease on glutathione-capped gold nanoparticles.

Science.gov (United States)

Garg, Seema; De, Arnab; Mozumdar, Subho

2015-05-01

Urease is a nickel-dependent metalloenzyme that catalyzes the hydrolysis of urea to form ammonia and carbon dioxide. Although the enzyme serves a significant role in several detoxification and analytical processes, its usability is restricted due to high cost, availability in small amounts, instability, and a limited possibility of economic recovery from a reaction mixture. Hence, there is a need to develop an efficient, simple, and reliable immobilization strategy for the enzyme. In this study, the carboxyl terminated surface of glutathione-capped gold nanoparticles have been utilized as a solid support for the covalent attachment of urease. The immobilization has been carried out at different pH conditions so as to elucidate its effect on the immobilization efficiency and enzyme bioactivity. The binding of the enzyme has been quantitatively and qualitatively analyzed through techniques like ultraviolet-visible spectroscopy, intrinsic steady state fluorescence, and circular dichorism. The bioactivity of the immobilized enzyme was investigated with respect to the native enzyme under different thermal conditions. Recyclability and shelf life studies of the immobilized enzyme have also been carried out. Results reveal that the immobilization is most effective at pH of 7.4 followed by that in an acidic medium and is least in alkaline environment. The immobilized enzyme also exhibits enhance activity in comparison to the native form at physiological temperature. The immobilized urease (on gold glutathione nanoconjugates surface) can be effectively employed for biosensor fabrication, immunoassays and as an in vivo diagnostic tool in the future. © 2014 Wiley Periodicals, Inc.

Immobilized Trienzymatic System with Enhanced Stabilization for the Biotransformation of Lactose

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Pedro Torres

2017-02-01

Full Text Available The use of ketohexose isomerases is a powerful tool in lactose whey processing, but these enzymes can be very sensitive and expensive. Development of immobilized/stabilized biocatalysts could be a further option to improve the process. In this work, β-galactosidase from Bacillus circulans, l-arabinose (d-galactose isomerase from Enterococcus faecium, and d-xylose (d-glucose isomerase from Streptomyces rubiginosus were immobilized individually onto Eupergit C and Eupergit C 250 L. Immobilized activity yields were over 90% in all cases. With the purpose of increasing thermostability of derivatives, two post-immobilization treatments were performed: alkaline incubation to favor the formation of additional covalent linkages, and blocking of excess oxirane groups by reacting with glycine. The greatest thermostability was achieved when alkaline incubation was carried out for 24 h, producing l-arabinose isomerase-Eupergit C derivatives with a half-life of 379 h and d-xylose isomerase-Eupergit C derivatives with a half-life of 554 h at 50 °C. Preliminary assays using immobilized and stabilized biocatalysts sequentially to biotransform lactose at pH 7.0 and 50 °C demonstrated improved performances as compared with soluble enzymes. Further improvements in ketohexose productivities were achieved when the three single-immobilizates were incubated simultaneously with lactose in a mono-reactor system.

Construction of an Immobilized Thermophilic Esterase on Epoxy Support for Poly(ε-caprolactone) Synthesis.

Science.gov (United States)

Ren, Hui; Xing, Zhen; Yang, Jiebing; Jiang, Wei; Zhang, Gang; Tang, Jun; Li, Quanshun

2016-06-18

Developing an efficient immobilized enzyme is of great significance for improving the operational stability of enzymes in poly(ε-caprolactone) synthesis. In this paper, a thermophilic esterase AFEST from the archaeon Archaeoglobus fulgidus was successfully immobilized on the epoxy support Sepabeads EC-EP via covalent attachment, and the immobilized enzyme was then employed as a biocatalyst for poly(ε-caprolactone) synthesis. The enzyme loading and recovered activity of immobilized enzyme was measured to be 72 mg/g and 10.4 U/mg using p-nitrophenyl caprylate as the substrate at 80 °C, respectively. Through the optimization of reaction conditions (enzyme concentration, temperature, reaction time and medium), poly(ε-caprolactone) was obtained with 100% monomer conversion and low number-average molecular weight (Mn enzyme exhibited excellent reusability, with monomer conversion values exceeding 75% during 15 batch reactions. Finally, poly(ε-caprolactone) was enzymatically synthesized with an isolated yield of 75% and Mn value of 3005 g/mol in a gram-scale reaction.

New technology for regiospecific covalent coupling of polysaccharide antigens in ELISA for serological detection

DEFF Research Database (Denmark)

Jauho, E.S.; Boas, Ulrik; Wiuff, Camilla

2000-01-01

In this study we demonstrate a new UV irradiation technique for covalent coupling of bacterial polysaccharides derived from lipopolysaccharides to microtiter plates and the use of such plates in an enzyme linked immunosorbent assay (ELISA). Lipopolysaccharides were cleaved by mild acid hydrolysis...... describe the use of this technique for the immobilization of Lipopolysaccharide derived polysaccharides from Salmonella Typhimurium and Salmonella Choleraesuis lipopolysaccharides, representing the O-antigens 1, 4, 5, 6, 7, and 12, The functional polysaccharide surface gave similar ELISA results to plates...

Phosphopeptide Enrichment by Covalent Chromatography after Derivatization of Protein Digests Immobilized on Reversed-Phase Supports

Science.gov (United States)

Nika, Heinz; Nieves, Edward; Hawke, David H.; Angeletti, Ruth Hogue

2013-01-01

A rugged sample-preparation method for comprehensive affinity enrichment of phosphopeptides from protein digests has been developed. The method uses a series of chemical reactions to incorporate efficiently and specifically a thiol-functionalized affinity tag into the analyte by barium hydroxide catalyzed β-elimination with Michael addition using 2-aminoethanethiol as nucleophile and subsequent thiolation of the resulting amino group with sulfosuccinimidyl-2-(biotinamido) ethyl-1,3-dithiopropionate. Gentle oxidation of cysteine residues, followed by acetylation of α- and ε-amino groups before these reactions, ensured selectivity of reversible capture of the modified phosphopeptides by covalent chromatography on activated thiol sepharose. The use of C18 reversed-phase supports as a miniaturized reaction bed facilitated optimization of the individual modification steps for throughput and completeness of derivatization. Reagents were exchanged directly on the supports, eliminating sample transfer between the reaction steps and thus, allowing the immobilized analyte to be carried through the multistep reaction scheme with minimal sample loss. The use of this sample-preparation method for phosphopeptide enrichment was demonstrated with low-level amounts of in-gel-digested protein. As applied to tryptic digests of α-S1- and β-casein, the method enabled the enrichment and detection of the phosphorylated peptides contained in the mixture, including the tetraphosphorylated species of β-casein, which has escaped chemical procedures reported previously. The isolates proved highly suitable for mapping the sites of phosphorylation by collisionally induced dissociation. β-Elimination, with consecutive Michael addition, expanded the use of the solid-phase-based enrichment strategy to phosphothreonyl peptides and to phosphoseryl/phosphothreonyl peptides derived from proline-directed kinase substrates and to their O-sulfono- and O-linked β-N-acetylglucosamine (O

Optimization of Phospholipase A1 Immobilization on Plasma Surface Modified Chitosan Nanofibrous Mat

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Zahra Beig Mohammadi

2016-01-01

Full Text Available Phospholipase A1 is known as an effective catalyst for hydrolysis of various phospholipids in enzymatic vegetable oil degumming. Immobilization is one of the most efficient strategies to improve its activity, recovery and functional properties. In this study, chitosan-co-polyethylene oxide (90:10 nanofibrous mat was successfully fabricated and modified with atmospheric plasma at different times (2, 6 and 10 min to interact with enzyme molecules. Scanning electron microscopy images revealed that the membranes retained uniform nanofibrous and open porous structures before and after the treatment. PLA1 was successfully immobilized onto the membrane surfaces via covalent bonds with the functional groups of chitosan nanofibrous mat. Response surface methodology was used to optimize the immobilization conditions for reaching the maximum immobilization efficiency. Enzyme concentration, pH, and immobilization time were found to be significant key factors. Under optimum conditions (5.03 h, pH 5.63, and enzyme dosage 654.36 UI, the atmospheric plasma surface modified chitosan nanofibers reached the highest immobilization efficiency (78.50%. Fourier transform infrared spectroscopy of the control and plasma surface-modified chitosan nanofibers revealed the functional groups of nanofibers and their reaction with the enzyme. The results indicated that surface modification by atmospheric plasma induced an increase in PLA1 loading on the membrane surfaces.

Protein immobilization on the surface of liposomes via carbodiimide activation in the presence of N-hydroxysulfosuccinimide.

Science.gov (United States)

Bogdanov, A A; Klibanov, A L; Torchilin, V P

1988-04-25

A method of the covalent immobilization of proteins on the surface of liposomes, containing 10% (by mol) of N-glutaryl phosphatidylethanolamine, is described. Carboxylic groups of liposomal N-glutaryl phosphatidylethanolamine were activated in the presence of water-soluble carbodiimide and N-hydroxysulfosuccinimide and reacted subsequently with protein amino groups. The liposome-protein conjugates formed contained up to 5 x 10(-4) mol protein/mol lipid. Lectins (RCA1 and WGA) upon immobilization on liposomes retained saccharide specificity and the ability to agglutinate red blood cells. The immobilization of mouse monoclonal IgG in a ratio of 3.5 x 10(-4) mol IgG/mol lipid was achieved. The liposome activation in the absence of N-hydroxysulfosuccinimide resulted in a 2-fold decrease of protein coupling yields.

Immobilization of trypsin on miniature incandescent bulbs for infrared-assisted proteolysis

Energy Technology Data Exchange (ETDEWEB)

Ge, Huimin; Bao, Huimin; Zhang, Luyan; Chen, Gang, E-mail: gangchen@fudan.edu.cn

2014-10-03

Highlights: • Trypsin was immobilized on miniature incandescent bulbs via chitosan coating. • The bulbs acted as enzymatic reactors and the generators of infrared radiation. • The bulb bioreactors were successfully employed in infrared-assisted proteolysis. • The proteolysis could accomplish within 5 min with high sequence coverages. - Abstract: A novel efficient proteolysis approach was developed based on trypsin-immobilized miniature incandescent bulbs and infrared (IR) radiation. Trypsin was covalently immobilized in the chitosan coating on the outer surface of miniature incandescent bulbs with the aid of glutaraldehyde. When an illuminated enzyme-immobilized bulb was immersed in protein solution, the emitted IR radiation could trigger and accelerate heterogeneous protein digestion. The feasibility and performance of the novel proteolysis approach were demonstrated by the digestion of hemoglobin (HEM), cytochrome c (Cyt-c), lysozyme (LYS), and ovalbumin (OVA) and the digestion time was significantly reduced to 5 min. The obtained digests were identified by MALDI-TOF-MS with the sequence coverages of 91%, 77%, 80%, and 52% for HEM, Cyt-c, LYS, and OVA (200 ng μL{sup −1} each), respectively. The suitability of the prepared bulb bioreactors to complex proteins was demonstrated by digesting human serum.

Dynamic covalent gels assembled from small molecules:from discrete gelators to dynamic covalent polymers

Institute of Scientific and Technical Information of China (English)

Jian-Yong Zhang; Li-Hua Zeng; Juan Feng

2017-01-01

Dynamic covalent chemistry has emerged recently to be a powerful tool to construct functional materials.This article reviews the progress in the research and development of dynamic covalent chemistry in gels assembled from small molecules.First dynamic covalent reactions used in gels are reviewed to understand the dynamic covalent bonding.Afterwards the catalogues of dynamic covalent gels are reviewed according to the nature of gelators and the interactions between gelators.Dynamic covalent bonding can be involved to form low molecular weight gelators.Low molecular weight molecules with multiple functional groups react to form dynamic covalent cross-linked polymers and act as gelators.Two catalogues of gels show different properties arising from their different structures.This review aims to illustrate the structure-property relationships of these dynamic covalent gels.

Immobilization of small molecules and proteins by radio-derivatized polystyrene

International Nuclear Information System (INIS)

Varga, J.M.; Fritsch, P.

1990-01-01

When molded polystyrene (PS) products (e.g., microtiter plates) or latex particles are irradiated with high-energy (1-10 Mrads) gamma rays in the presence of nonpolymerizable small molecules such as aromatic amines, some of these molecules incorporate into PS, which leads to the formation of radio-derivatized PS (RDPS). Two classes of RDPS can be identified regarding their ability for immobilization of biologically important molecules: (1) reactive RDPS that are able to form covalent bonds with molecules such as proteins without the help of cross-linkers, and (2) functionalized RDPS that can be used for the immobilization of molecules with activators (e.g., carbodiimides) or cross-linkers. The method can be used for the production of low-noise supports for binding assays. Most of the RDPS can be produced without impairment of the optical quality of PS, making derivatized microtiter plates suitable for colorimetric assays. The principle can be applied for the preparation of affinity sorbents, e.g., for high-performance affinity chromatography and for the immobilization of enzymes using latex PS particles

Tailored functionalization of iron oxide nanoparticles for MRI, drug delivery, magnetic separation and immobilization of biosubstances.

Science.gov (United States)

Hola, Katerina; Markova, Zdenka; Zoppellaro, Giorgio; Tucek, Jiri; Zboril, Radek

2015-11-01

In this critical review, we outline various covalent and non-covalent approaches for the functionalization of iron oxide nanoparticles (IONPs). Tuning the surface chemistry and design of magnetic nanoparticles are described in relation to their applicability in advanced medical technologies and biotechnologies including magnetic resonance imaging (MRI) contrast agents, targeted drug delivery, magnetic separations and immobilizations of proteins, enzymes, antibodies, targeting agents and other biosubstances. We review synthetic strategies for the controlled preparation of IONPs modified with frequently used functional groups including amine, carboxyl and hydroxyl groups as well as the preparation of IONPs functionalized with other species, e.g., epoxy, thiol, alkane, azide, and alkyne groups. Three main coupling strategies for linking IONPs with active agents are presented: (i) chemical modification of amine groups on the surface of IONPs, (ii) chemical modification of bioactive substances (e.g. with fluorescent dyes), and (iii) the activation of carboxyl groups mainly for enzyme immobilization. Applications for drug delivery using click chemistry linking or biodegradable bonds are compared to non-covalent methods based on polymer modified condensed magnetic nanoclusters. Among many challenges, we highlight the specific surface engineering allowing both therapeutic and diagnostic applications (theranostics) of IONPs and magnetic/metallic hybrid nanostructures possessing a huge potential in biocatalysis, green chemistry, magnetic bioseparations and bioimaging. Copyright © 2015 Elsevier Inc. All rights reserved.

Layer by layer assembly of a biocatalytic packaging film: lactase covalently bound to low-density polyethylene.

Science.gov (United States)

Wong, Dana E; Talbert, Joey N; Goddard, Julie M

2013-06-01

Active packaging is utilized to overcome limitations of traditional processing to enhance the health, safety, economics, and shelf life of foods. Active packaging employs active components to interact with food constituents to give a desired effect. Herein we describe the development of an active package in which lactase is covalently attached to low-density polyethylene (LDPE) for in-package production of lactose-free dairy products. The specific goal of this work is to increase the total protein content loading onto LDPE using layer by layer (LbL) deposition, alternating polyethylenimine, glutaraldehyde (GL), and lactase, to enhance the overall activity of covalently attached lactase. The films were successfully oxidized via ultraviolet light, functionalized with polyethylenimine and glutaraldehyde, and layered with immobilized purified lactase. The total protein content increased with each additional layer of conjugated lactase, the 5-layer sample reaching up to 1.3 μg/cm2 . However, the increase in total protein did not lend to an increase in overall lactase activity. Calculated apparent Km indicated the affinity of immobilized lactase to substrate remains unchanged when compared to free lactase. Calculated apparent turnover numbers (kcat ) showed with each layer of attached lactase, a decrease in substrate turnover was experienced when compared to free lactase; with a decrease from 128.43 to 4.76 s(-1) for a 5-layer conjugation. Our results indicate that while LbL attachment of lactase to LDPE successfully increases total protein mass of the bulk material, the adverse impact in enzyme efficiency may limit the application of LbL immobilization chemistry for bioactive packaging use. © 2013 Institute of Food Technologists®

Immobilization and controlled release of drug using plasma polymerized thin film

Energy Technology Data Exchange (ETDEWEB)

Myung, Sung-Woon [Department of Dental Materials, School of Dentistry, MRC Center, Chosun University, 309 Pilmun-daero, Dong-gu, Gwangju (Korea, Republic of); Jung, Sang-Chul [Department of Environmental Engineering, Sunchon National University, Sunchon 540-742 (Korea, Republic of); Kim, Byung-Hoon, E-mail: kim5055@chosun.ac.kr [Department of Dental Materials, School of Dentistry, MRC Center, Chosun University, 309 Pilmun-daero, Dong-gu, Gwangju (Korea, Republic of)

2015-06-01

In this study, plasma polymerization of acrylic acid was employed to immobilize drug and control its release. Doxorubicin (DOX) was immobilized covalently on the glass surface deposited with plasma polymerized acrylic acid (PPAAc) thin film containing the carboxylic group. At first, the PPAAc thin film was coated on a glass surface at a pressure of 1.33 Pa and radio frequency (RF) discharge power of 20 W for 10 min. DOX was immobilized on the PPAAc deposition in a two environment of phosphate buffer saline (PBS) and dimethyl sulfoxide (DMSO) solutions. The DOX immobilized surface was characterized by scanning electron microscope, atomic force microscope and attenuated total reflection Fourier transform infrared spectroscopy. The DOX molecules were more immobilized in PBS than DMSO solution. The different immobilization and release profiles of DOX result from the solubility of hydrophobic DOX in aqueous and organic solutions. Second, in order to control the release of the drug, PPAAc thin film was covered over DOX dispersed layer. Different thicknesses and cross-linked PPAAc thin films by adjusting deposition time and RF discharge power were covered on the DOX layer dispersed. PPAAc thin film coated DOX layer reduced the release rate of DOX. The thickness control of plasma deposition allows controlling the release rate of drug. - Highlights: • Doxorubicin was immobilized on the surface of plasma polymerized acrylic acid thin film. • Release profile of doxorubicin was affected by aqueous and organic solutions. • Plasma polymerized acrylic acid thin film can be used to achieve controlled release.

Immobilization and controlled release of drug using plasma polymerized thin film

International Nuclear Information System (INIS)

Myung, Sung-Woon; Jung, Sang-Chul; Kim, Byung-Hoon

2015-01-01

In this study, plasma polymerization of acrylic acid was employed to immobilize drug and control its release. Doxorubicin (DOX) was immobilized covalently on the glass surface deposited with plasma polymerized acrylic acid (PPAAc) thin film containing the carboxylic group. At first, the PPAAc thin film was coated on a glass surface at a pressure of 1.33 Pa and radio frequency (RF) discharge power of 20 W for 10 min. DOX was immobilized on the PPAAc deposition in a two environment of phosphate buffer saline (PBS) and dimethyl sulfoxide (DMSO) solutions. The DOX immobilized surface was characterized by scanning electron microscope, atomic force microscope and attenuated total reflection Fourier transform infrared spectroscopy. The DOX molecules were more immobilized in PBS than DMSO solution. The different immobilization and release profiles of DOX result from the solubility of hydrophobic DOX in aqueous and organic solutions. Second, in order to control the release of the drug, PPAAc thin film was covered over DOX dispersed layer. Different thicknesses and cross-linked PPAAc thin films by adjusting deposition time and RF discharge power were covered on the DOX layer dispersed. PPAAc thin film coated DOX layer reduced the release rate of DOX. The thickness control of plasma deposition allows controlling the release rate of drug. - Highlights: • Doxorubicin was immobilized on the surface of plasma polymerized acrylic acid thin film. • Release profile of doxorubicin was affected by aqueous and organic solutions. • Plasma polymerized acrylic acid thin film can be used to achieve controlled release

Direct Profiling the Post-Translational Modification Codes of a Single Protein Immobilized on a Surface Using Cu-free Click Chemistry.

Science.gov (United States)

Kim, Kyung Lock; Park, Kyeng Min; Murray, James; Kim, Kimoon; Ryu, Sung Ho

2018-05-23

Combinatorial post-translational modifications (PTMs), which can serve as dynamic "molecular barcodes", have been proposed to regulate distinct protein functions. However, studies of combinatorial PTMs on single protein molecules have been hindered by a lack of suitable analytical methods. Here, we describe erasable single-molecule blotting (eSiMBlot) for combinatorial PTM profiling. This assay is performed in a highly multiplexed manner and leverages the benefits of covalent protein immobilization, cyclic probing with different antibodies, and single molecule fluorescence imaging. Especially, facile and efficient covalent immobilization on a surface using Cu-free click chemistry permits multiple rounds (>10) of antibody erasing/reprobing without loss of antigenicity. Moreover, cumulative detection of coregistered multiple data sets for immobilized single-epitope molecules, such as HA peptide, can be used to increase the antibody detection rate. Finally, eSiMBlot enables direct visualization and quantitative profiling of combinatorial PTM codes at the single-molecule level, as we demonstrate by revealing the novel phospho-codes of ligand-induced epidermal growth factor receptor. Thus, eSiMBlot provides an unprecedentedly simple, rapid, and versatile platform for analyzing the vast number of combinatorial PTMs in biological pathways.

Sodium bicarbonate-gelled chitosan beads as mechanically stable carriers for the covalent immobilization of enzymes.

Science.gov (United States)

Wahba, Marwa I

2018-03-01

The poor mechanical stability of chitosan has long impeded its industrial utilization as an immobilization carrier. In this study, the mechanical properties of chitosan beads were greatly improved through utilizing the slow rate of the sodium bicarbonate-induced chitosan gelation and combining it with the chemical cross-linking action of glutaraldehyde (GA). The GA-treated sodium bicarbonate-gelled chitosan beads exhibited much better mechanical properties and up to 2.45-fold higher observed activity of the immobilized enzyme (β-D-galactosidase (β-gal)) when compared to the GA-treated sodium tripolyphosphate (TPP)-gelled chitosan beads. The differences between the sodium bicarbonate-gelled and the TPP-gelled chitosan beads were proven visually and also via scanning electron microscopy, elemental analysis, and differential scanning calorimetry. Moreover, the optimum pH, the optimum temperature, the apparent K m , and the apparent V max of the β-gals immobilized onto the two aforementioned types of chitosan beads were determined and compared. A reusability study was also performed. This study proved the superiority of the sodium bicarbonate-gelled chitosan beads as they retained 72.22 ± 4.57% of their initial observed activity during the 13 th reusability cycle whereas the TPP-gelled beads lost their activity during the first four reusability cycles, owing to their fragmentation. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:347-361, 2018. © 2017 American Institute of Chemical Engineers.

Immobilization of surface active compounds on polymer supports using glow discharge processess. 1. Sodium dodecyl sulfate on poly(propylene)

NARCIS (Netherlands)

Terlingen, J.G.A.; Terlingen, Johannes G.A.; Feijen, Jan; Hoffman, Allan S.

1993-01-01

A new method has been developed in which a reversibly adsorbed layer of a surfactant (sodium dodecyl sulfate, SDS) is covalently immobilized in one step onto a hydrophobic substrate (poly(propylene), PP) by applying an argon plasma treatment. The adsorption of SDS from aqueous solutions onto PP

Produção de concentrados de frutose por inulinases de Penicillium janczewskii e atividade sobre o nível de glicose plasmática em ratos diabéticos Fructose syrups produced by inulinases from Penicillium janczewskii and activity on plasma glucose level in diabetic rats

Directory of Open Access Journals (Sweden)

Rosemeire A. Bom Pessoni

2004-09-01

Full Text Available A frutose é utilizada atualmente como adoçante para diabéticos, sendo produzida comercialmente por hidrólise do amido, sob um processo de alto custo que envolve três etapas enzimáticas usando alfa-amilase, amiloglicosidase e glicose isomerase. Uma alternativa para a produção de concentrados de frutose é a hidrólise enzimática da inulina, polímero de frutose encontrado em Asteráceas, incluindo espécies nativas do cerrado. Nesse caso, através de uma única etapa enzimática obtêm-se concentrados com até 95% de frutose. Embora baixos níveis desse açúcar possam ser metabolizados na ausência de insulina, seu efeito sobre a redução do nível de glicose plasmática ainda não está completamente esclarecido. No presente trabalho foi avaliada a ação da frutose produzida por hidrólise da inulina de Vernonia herbacea (Asteraceae por inulinases de Penicillium janczewskii no nível de glicose plasmática de ratos diabéticos. Dentre os animais diabéticos tratados não foi verificada mortalidade, havendo redução de 46% em média (pFructose has been used as sweetener by patients with diabetes. This sugar is usually produced from starch by a high-cost enzymatic process, which envolves the utilization of alpha-amylase, amyloglucosidase and glucose isomerase. Fructose can be alternatively produced by the enzymatic hydrolysis of inulin, a polymer of fructose stored as reserve in a number of Asteraceae species. Using only one enzymatic step, inulin can be converted into syrups containing up to 95% fructose. In the present work, fructose syrup was produced from inulin of Vernonia herbacea by hydrolysis with extracellular inulinases from Penicillium janczewskii and evaluated with respect to the effect on plasma glucose level in diabetic rats. Reduction of ca. 46% (p<1% Tukey test of glucose levels in the plasma and no mortality were observed when rats were treated with hydrolysate of inulin. The high amounts of inulin stored by V

Immobilizing affinity proteins to nitrocellulose: a toolbox for paper-based assay developers.

Science.gov (United States)

Holstein, Carly A; Chevalier, Aaron; Bennett, Steven; Anderson, Caitlin E; Keniston, Karen; Olsen, Cathryn; Li, Bing; Bales, Brian; Moore, David R; Fu, Elain; Baker, David; Yager, Paul

2016-02-01

To enable enhanced paper-based diagnostics with improved detection capabilities, new methods are needed to immobilize affinity reagents to porous substrates, especially for capture molecules other than IgG. To this end, we have developed and characterized three novel methods for immobilizing protein-based affinity reagents to nitrocellulose membranes. We have demonstrated these methods using recombinant affinity proteins for the influenza surface protein hemagglutinin, leveraging the customizability of these recombinant "flu binders" for the design of features for immobilization. The three approaches shown are: (1) covalent attachment of thiolated affinity protein to an epoxide-functionalized nitrocellulose membrane, (2) attachment of biotinylated affinity protein through a nitrocellulose-binding streptavidin anchor protein, and (3) fusion of affinity protein to a novel nitrocellulose-binding anchor protein for direct coupling and immobilization. We also characterized the use of direct adsorption for the flu binders, as a point of comparison and motivation for these novel methods. Finally, we demonstrated that these novel methods can provide improved performance to an influenza hemagglutinin assay, compared to a traditional antibody-based capture system. Taken together, this work advances the toolkit available for the development of next-generation paper-based diagnostics.

Biofunctional Paper via Covalent Modification of Cellulose

Science.gov (United States)

Yu, Arthur; Shang, Jing; Cheng, Fang; Paik, Bradford A.; Kaplan, Justin M.; Andrade, Rodrigo B.; Ratner, Daniel M.

2012-01-01

Paper-based analytical devices are the subject of growing interest for the development of low-cost point-of-care diagnostics, environmental monitoring technologies and research tools for limited-resource settings. However, there are limited chemistries available for the conjugation of biomolecules to cellulose for use in biomedical applications. Herein, divinyl sulfone (DVS) chemistry was demonstrated to covalently immobilize small molecules, proteins and DNA onto the hydroxyl groups of cellulose membranes through nucleophilic addition. Assays on modified cellulose using protein-carbohydrate and protein-glycoprotein interactions as well as oligonucleotide hybridization showed that the membrane’s bioactivity was specific, dose-dependent, and stable over a long period of time. Use of an inkjet printer to form patterns of biomolecules on DVS-activated cellulose illustrates the adaptability of the DVS functionalization technique to pattern sophisticated designs, with potential applications in cellulose-based lateral flow devices. PMID:22708701

Fabrication and characterization of all-covalent nanocomposite functionalized screen-printed voltammetric sensors

International Nuclear Information System (INIS)

Jasmin, Jean-Philippe; Cannizzo, Caroline; Dumas, Eddy; Chaussé, Annie

2014-01-01

Highlights: • Screen printed electrodes were covalently functionalized by gold nanoparticles. • The covalent grafting of AuNPs was achieved via diazonium salt chemistry. • Two grafting methods and two types of AuNPs were compared. • Carboxylate ligands were grafted on these nanostructured electrodes. • Good preliminary responses towards lead analysis were obtained by SW-ASV. - Abstract: We report in this paper an all-covalent method to obtain highly nanostructured carbon screen printed electrodes (SPEs) bearing gold nanoparticles (AuNPs) functionalized by complexing groups using diazonium salts chemistry. SPEs were first modified with 4-aminophenyl functions (SPE-Ph-NH 2 ). The amino moieties were then converted into diazonium salts (SPE-Ph-N 2 + Cl − ). These reactive SPEs were then used to immobilize AuNPs by electrochemical or spontaneous method. The spontaneous method proved to be a more efficient grafting approach. Two types of AuNPs suspensions were compared: AuNPs obtained via the well-known Turkevich method, citrate-stabilized and having a diameter of about 20 nm, and AuNPs obtained by the method recently described by Eah et al., stabilizer-free with an average diameter of 4 nm. We show that the size of the Au-NPs, their concentration and their surface properties are key parameters that affect the electrochemical properties of the final nanostructured SPEs. The covalent grafting of 4-carboxyphenyl ligands through diazonium chemistry, able to complex metallic cations, at the surface of SPE-Ph-AuNPs allowed their use for the detection of Pb(II). Electrochemical Impedance Spectroscopy, Cyclic Voltammetry, Scanning Electron Microscopy, Rutherford Backscattering and X-ray Photoelectron Spectroscopy were used to characterize these nanostructured materials

Biofunctional paper via the covalent modification of cellulose.

Science.gov (United States)

Yu, Arthur; Shang, Jing; Cheng, Fang; Paik, Bradford A; Kaplan, Justin M; Andrade, Rodrigo B; Ratner, Daniel M

2012-07-31

Paper-based analytical devices are the subject of growing interest for the development of low-cost point-of-care diagnostics, environmental monitoring technologies, and research tools for limited-resource settings. However, there are limited chemistries available for the conjugation of biomolecules to cellulose for use in biomedical applications. Herein, divinyl sulfone (DVS) chemistry was demonstrated to immobilize small molecules, proteins, and DNA covalently onto the hydroxyl groups of cellulose membranes through nucleophilic addition. Assays on modified cellulose using protein-carbohydrate and protein-glycoprotein interactions as well as oligonucleotide hybridization showed that the membrane's bioactivity was specific, dose-dependent, and stable over a long period of time. The use of an inkjet printer to form patterns of biomolecules on DVS-activated cellulose illustrates the adaptability of the DVS functionalization technique to pattern sophisticated designs, with potential applications in cellulose-based lateral flow devices.

Immobilization of glucose oxidase on sepharose by UV-initiated graft copolymerization

International Nuclear Information System (INIS)

D'Angiuro, L.; Cremonesi, P.

1982-01-01

The performance of a new method of enzyme immobilization based on photochemically initiated direct graft copolymerization was recently investigated. The immobilization reaction can be carried out in a simple way and by carefully selecting the reaction conditions, the enzyme-graft copolymer can be obtained as the main reaction product. Coupling efficiency of glucose oxidase has been found to depend only on the amount of photocatalyst (FeCl 3 ) fixed on Sepharose used as polysaccharide support. Small quantities of glycidylmethacrylate (GMA) (0.25 g/g dry Sepharose) are sufficient but necessary to achieve the best enzyme coupling efficiency (20-40%). Enzyme immobilization occurs very rapidly and the entire reaction occurs within 60 min. Reaction patterns and physicochemical characteristics of the obtained enzyme-graft copolymers exclude the glucose oxidase entrapment: therefore a covalent attachment mechanism may be proposed. The kinetic parameters of immobilized glucose oxidase (K/sub m/' = 2.0 x 10 -2 M) are quite similar to those of free enzyme (K/sub m/ = 1.93 x 10 -2 M), and no diffusion limitation phenomena are evidenced in samples having different enzyme or polymer content. Lyophilization, thermostability, and long-term continuous operation also have been investigated. The advantages of this method over that using vinylenzyme copolymerization are discussed

Covalent co-immobilization of heparin/laminin complex that with different concentration ratio on titanium surface for selectively direction of platelets and vascular cells behavior

International Nuclear Information System (INIS)

Wang, Jian; Chen, Yuan; Liu, Tao; Wang, Xue; Liu, Yang; Wang, Yuan; Chen, Junying; Huang, Nan

2014-01-01

Highlights: • Extracellular matrix inspired surface modification with fibronectin, heparin and VEGF to construct a favorable microenvironment for selectively anticoagulant and promote endothelialization. • Take the advantage of specific intermolecular interaction, the bioactivity of above biomolecules was more efficiently maintained in compared with the common used covalent immobilization method. • Poly-l-lysine was used as a novel interlayer for surface amination, and in comparison, PLL coating was more feasible and the degradation product had no harm to human body. - Abstract: Surface biofunctional modification of coronary artery stent to improve the hemocompatibility and selectively accelerate endothelium regeneration but prevent restenosis have been become a new hotspot. For this, a novel method was developed in this work by co-immobilization of Ln and heparin complex on poly-L-lysine modified Ti surface. Take the advantage of the specific interaction between Ln and heparin, Ln and heparin complexes with different concentration ratios were set up for creating different exposure density of these two types of biomolecules. According to biocompatibility evaluation results, the Hep/Ln complexes modified surface displayed less platelet adhesion and activation. Especially, on L(150)H and L(200)H surface, the AT III binding quantity, APTT value and anti-coagulation property of modified surface were significantly promoted. Furthermore, the adherent density and proliferation activity of ECs and EPCs were positively correlated with Ln concentration. Notably, the proliferation of both ECs and EPCs on L(100)H, L(150)H and L(200)H surface were greatly promoted. Another hand, the proliferation activity of SMCs was significantly inhibited on Hep/Ln modified surfaces, which was considered mainly due to the inhibitory effect of heparin to SMCs. According to the existing results, this study demonstrated that in a certain range of heparin and laminin concentration ratio

Geometry-dependent DNA-TiO2 immobilization mechanism: A spectroscopic approach

Science.gov (United States)

Silva-Moraes, M. O.; Garcia-Basabe, Y.; de Souza, R. F. B.; Mota, A. J.; Passos, R. R.; Galante, D.; Fonseca Filho, H. D.; Romaguera-Barcelay, Y.; Rocco, M. L. M.; Brito, W. R.

2018-06-01

DNA nucleotides are used as a molecular recognition system on electrodes modified to be applied in the detection of various diseases, but immobilization mechanisms, as well as, charge transfers are not satisfactorily described in the literature. An electrochemical and spectroscopic study was carried out to characterize the molecular groups involved in the direct immobilization of DNA structures on the surface of nanostructured TiO2 with the aim of evaluating the influence of the geometrical aspects. X-ray photoelectron spectroscopy at O1s and P2p core levels indicate that immobilization of DNA samples occurs through covalent (Psbnd Osbnd Ti) bonds. X-ray absorption spectra at the Ti2p edge reinforce this conclusion. A new species at 138.5 eV was reported from P2p XPS spectra analysis which plays an important role in DNA-TiO2 immobilization. The Psbnd Osbnd Ti/Osbnd Ti ratio showed that quantitatively the DNA immobilization mechanism is dependent on their geometry, becoming more efficient for plasmid ds-DNA structures than for PCR ds-DNA structures. The analysis of photoabsorption spectra at C1s edge revealed that the molecular groups that participate in the C1s → LUMO electronic transitions have different pathways in the charge transfer processes at the DNA-TiO2 interface. Our results may contribute to additional studies of immobilization mechanisms understanding the influence of the geometry of different DNA molecules on nanostructured semiconductor and possible impact to the charge transfer processes with application in biosensors or aptamers.

Biodiesel production by using lipase immobilized onto novel silica-based hybrid foams

Energy Technology Data Exchange (ETDEWEB)

Brun, Nicolas [Centre de Recherche Paul Pascal, Pessac (France); Institut des Sciences Moleculaires, Talence (France); Garcia, Annick Babeau; Oestreicher, Victor; Durand, Fabien; Backov, Renal [Centre de Recherche Paul Pascal, Pessac (France); Deleuze, Herve [Institut des Sciences Moleculaires, Talence (France); Laurent, Guillaume; Sanchez, Clement [Laboratoire de Chimie de la Matiere Condensee, Paris (France)

2010-07-01

The covalent immobilization of crude lipases within silica-based macroporous frameworks have been performed by combining sol-gel process, concentrated direct emulsion, lyotropic mesophase and post-synthesis functionalizations. The assynthesized open cell hybrid monoliths exhibit high macroscopic porosity, around 90%, providing interconnected scaffold while reducing the diffusion low kinetic issue. The entrapment of enzymes in such foams deals with a high stability over esterification of fatty acids, hydrolysis of triglycerides (not shown herein) and biodiesel production by transesterification. (orig.)

Optical Biosensor with Multienzyme System Immobilized onto Hybrid Membrane for Pesticides Determination

Directory of Open Access Journals (Sweden)

Lyubov Yotova

2011-12-01

Full Text Available A construction of optical biosensor based on simultaneous immobilization of acetylcholinesterase and choline oxidase enzymes for the detection of pesticides residues is described. Different kinds of novel SiO2 hybrid membranes were synthesized to be suitable for optical biosensors using sol-gel techniques. The bioactive component of the sensor consists of a multi-enzyme system including acetylcholinesterase and choline oxidase covalently immobilized on new hybrid membranes. The sensor exhibited a linear response to acetylcholine in a concentration range of 2.5 - 30 mM. Inhibition plots obtained from testing carbamate (carbofuran pesticides exhibited concentration dependent behaviour and showed linear profiles in concentration ranges between 5x10-8 - 5x10-7 M for carbofuran. The factors affecting the constructed optical biosensors were investigated.

Enzymatic milk clotting activity in artichoke (Cynara scolymus) leaves and alpine thistle (Carduus defloratus) flowers. Immobilization of alpine thistle aspartic protease.

Science.gov (United States)

Esposito, Marilena; Di Pierro, Prospero; Dejonghe, Winnie; Mariniello, Loredana; Porta, Raffaele

2016-08-01

Two different milk clotting enzymes, belonging to the aspartic protease family, were extracted from both artichoke leaves and alpine thistle flowers, and the latter was covalently immobilized by using a polyacrylic support containing polar epoxy groups. Our findings showed that the alpine thistle aspartic protease was successfully immobilized at pH 7.0 on Immobeads IB-150P beads and that, under these experimental conditions, an immobilization yield of about 68% and a recovery of about 54% were obtained. Since the enzyme showed an optimal pH of 5.0, a value very similar to the one generally used for milk clotting during cheese making, and exhibited a satisfactory stability over time, the use of such immobilized vegetable rennet for the production of novel dairy products is suggested. Copyright © 2016. Published by Elsevier Ltd.

Impedimetric Urea Biosensor Based on Modified Gold Electrode with Urease Immobilized on Glutathione Layer

OpenAIRE

Houcine BARHOUMI; Abderrazak MAAREF; Nicole JAFFREZIC-RENAULT

2014-01-01

In this work, a glutathione (GSH) modified gold microelectrode was used for the covalent immobilization of urease biomolecules via the glutaraldehyde-coupling agent. The self- assembled monolayers (SAMs) onto the gold surface was investigated by using the electrochemical impedance spectroscopy measurements (EIS). Before urease grafting, a significant interaction was noticed between urea and the glutathione layer by forming hydrogen bonds. The H-NMR analysis was carried out to highlight the po...

Magnetic nanoparticles coated with polyaniline to stabilize immobilized trypsin

Energy Technology Data Exchange (ETDEWEB)

Maciel, J. C., E-mail: jackeline-maciel@hotmail.com [Universidade Federal de Roraima (Brazil); Mercês, A. A. D.; Cabrera, M. [Universidade Federal de Pernambuco, Laboratório de Imunopatologia Keizo Asami (Brazil); Shigeyosi, W. T. [Universidade Federal de São Carlos, Departamento de Física (Brazil); Souza, S. D. de; Olzon-Dionysio, M.; Fabris, J. D. [Universidade Federal dos Vales de Jequitinhonha e Mucuri (Brazil); Cardoso, C. A. [Universidade Federal de São Carlos, Departamento de Física (Brazil); Neri, D. F. M. [Universidade Federal do Vale do São Francisco (Brazil); Silva, M. P. C.; Carvalho, L. B. [Universidade Federal de Pernambuco, Laboratório de Imunopatologia Keizo Asami (Brazil)

2016-12-15

It is reported the synthesis of magnetic nanoparticles via the chemical co-precipitation of Fe {sup 3+} ions and their preparation by coating them with polyaniline. The electronic micrograph analysis showed that the mean diameter for the nanoparticles is ∼15 nm. FTIR, powder X-ray diffraction and Mössbauer spectroscopy were used to understand the chemical, crystallographic and {sup 57}Fe hyperfine structures for the two samples. The nanoparticles, which exhibited magnetic behavior with relatively high spontaneous magnetization at room temperature, were identified as being mainly formed by maghemite (γFe{sub 2}O{sub 3}). The coated magnetic nanoparticles (sample labeled “mPANI”) presented a real ability to bind biological molecules such as trypsin, forming the magnetic enzyme derivative (sample “mPANIG-Trypsin”). The amount of protein and specific activity of the immobilized trypsin were found to be 13±5 μg of protein/mg of mPANI (49.3 % of immobilized protein) and 24.1±0.7 U/mg of immobilized protein, respectively. After 48 days of storage at 4 {sup ∘}C, the activity of the immobilized trypsin was found to be 89 % of its initial activity. This simple, fast and low-cost procedure was revealed to be a promising way to prepare mPANI nanoparticles if technological applications addressed to covalently link biomolecules are envisaged. This route yields chemically stable derivatives, which can be easily recovered from the reaction mixture with a magnetic field and recyclable reused.

Immobilization of alcohol dehydrogenase on ceramic silicon carbide membranes for enzymatic CH3 OH production

DEFF Research Database (Denmark)

Zeuner, Birgitte; Ma, Nicolaj; Berendt, Kasper

2018-01-01

BACKGROUND Alcohol dehydrogenase (ADH; EC 1.1.1.1) catalyzes oxidation of CH3OH to CHOH during NAD+ reduction to NADH. ADH can also accelerate the reverse reaction, which is studied as part of cascadic enzymatic conversion of CO2 to CH3OH. In the present study, immobilization of ADH onto macropor......BACKGROUND Alcohol dehydrogenase (ADH; EC 1.1.1.1) catalyzes oxidation of CH3OH to CHOH during NAD+ reduction to NADH. ADH can also accelerate the reverse reaction, which is studied as part of cascadic enzymatic conversion of CO2 to CH3OH. In the present study, immobilization of ADH onto......‐of‐concept for the use of NaOH‐treated SiC membranes for covalent enzyme immobilization and biocatalytic efficiency improvement of ADH during multiple reaction cycles. These data have implications for the development of robust extended enzymatic reactions....

Plasma Treated High-Density Polyethylene (HDPE Medpor Implant Immobilized with rhBMP-2 for Improving the Bone Regeneration

Directory of Open Access Journals (Sweden)

Jin-Su Lim

2014-01-01

Full Text Available We investigate the bone generation capacity of recombinant human bone morphogenetic protein-2 (rhBMP-2 immobilized Medpor surface through acrylic acid plasma-polymerization. Plasma-polymerization was carried out at a 20 W at an acrylic acid flow rate of 7 sccm for 5 min. The plasma-polymerized Medpor surface showed hydrophilic properties and possessed a high density of carboxyl groups. The rhBMP-2 was immobilized with covalently attached carboxyl groups using 1-ethyl-3-(3-dimethylaminopropyl carbodiimide and N-hydroxysuccinimide. Carboxyl groups and rhBMP-2 immobilization on the Medpor surface were identified by Fourier transform infrared spectroscopy. The activity of Medpor with rhBMP-2 immobilized was examined using an alkaline phosphatase assay on MC3T3-E1 cultured Medpor. These results showed that the rhBMP-2 immobilized Medpor increased the level of MC3T3-E1 cell differentiation. These results demonstrated that plasma surface modification has the potential to immobilize rhBMP-2 on polymer implant such as Medpor and can be used for the binding of bioactive nanomolecules in bone tissue engineering.

Immobilization of α-Amylase from Anoxybacillus sp. SK3-4 on ReliZyme and Immobead Supports

Directory of Open Access Journals (Sweden)

Ummirul Mukminin Kahar

2016-09-01

Full Text Available α-Amylase from Anoxybacillus sp. SK3-4 (ASKA is a thermostable enzyme that produces a high level of maltose from starches. A truncated ASKA (TASKA variant with improved expression and purification efficiency was characterized in an earlier study. In this work, TASKA was purified and immobilized through covalent attachment on three epoxide (ReliZyme EP403/M, Immobead IB-150P, and Immobead IB-150A and an amino-epoxide (ReliZyme HFA403/M activated supports. Several parameters affecting immobilization were analyzed, including the pH, temperature, and quantity (mg of enzyme added per gram of support. The influence of the carrier surface properties, pore sizes, and lengths of spacer arms (functional groups on biocatalyst performances were studied. Free and immobilized TASKAs were stable at pH 6.0–9.0 and active at pH 8.0. The enzyme showed optimal activity and considerable stability at 60 °C. Immobilized TASKA retained 50% of its initial activity after 5–12 cycles of reuse. Upon degradation of starches and amylose, only immobilized TASKA on ReliZyme HFA403/M has comparable hydrolytic ability with the free enzyme. To the best of our knowledge, this is the first report of an immobilization study of an α-amylase from Anoxybacillus spp. and the first report of α-amylase immobilization using ReliZyme and Immobeads as supports.

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

International Nuclear Information System (INIS)

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

2010-01-01

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

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

Energy Technology Data Exchange (ETDEWEB)

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

2010-07-15

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

Covalent modification of boron-doped diamond electrodes with an imidazolium-based ionic liquid

Energy Technology Data Exchange (ETDEWEB)

Wang Mei [Institut de Recherche Interdisciplinaire (IRI, USR 3078), Parc de la Haute Borne, 50 Avenue de Halley, BP 70478, 59658 Villeneuve d' Ascq (France); Institut d' Electronique, de Microelectronique et de Nanotechnologie (IEMN, UMR 8520), Cite Scientifique, Avenue Poincare, BP 60069, 59652 Villeneuve d' Ascq (France); School of Materials Science and Engineering, Shandong University, 19723 Jingshi Road, Jinan, Shandong Province (China); Schneider, Amene [Austrian Centre of Competence for Tribology, Viktor Kaplan Strasse 2, 2700, Wiener Neustadt (Austria); Niedziolka-Joensson, Joanna; Marcon, Lionel [Institut de Recherche Interdisciplinaire (IRI, USR 3078), Parc de la Haute Borne, 50 Avenue de Halley, BP 70478, 59658 Villeneuve d' Ascq (France); Institut d' Electronique, de Microelectronique et de Nanotechnologie (IEMN, UMR 8520), Cite Scientifique, Avenue Poincare, BP 60069, 59652 Villeneuve d' Ascq (France); Ghodbane, Slimane; Steinmueller-Nethl, Doris [Rho-BeSt Coating GmbH, Exlgasse 20a, 6020 Innsbruck (Austria); Li Musen [School of Materials Science and Engineering, Shandong University, 19723 Jingshi Road, Jinan, Shandong Province (China); Boukherroub, Rabah [Institut de Recherche Interdisciplinaire (IRI, USR 3078), Parc de la Haute Borne, 50 Avenue de Halley, BP 70478, 59658 Villeneuve d' Ascq (France); Institut d' Electronique, de Microelectronique et de Nanotechnologie (IEMN, UMR 8520), Cite Scientifique, Avenue Poincare, BP 60069, 59652 Villeneuve d' Ascq (France); Szunerits, Sabine, E-mail: sabine.szunerits@iri.univ-lille1.f [Institut de Recherche Interdisciplinaire (IRI, USR 3078), Parc de la Haute Borne, 50 Avenue de Halley, BP 70478, 59658 Villeneuve d' Ascq (France); Institut d' Electronique, de Microelectronique et de Nanotechnologie (IEMN, UMR 8520), Cite Scientifique, Avenue Poincare, BP 60069, 59652 Villeneuve d' Ascq (France)

2010-02-01

An ionic liquid (IL, 1-(methylcarboxylic acid)-3-octylimidazolium-bis (trifluoromethylsulfonyl)imide) was covalently coupled onto a boron-doped diamond (BDD) surface through an esterification reaction. The resulting surface was characterized by X-ray photoelectron spectroscopy, water contact angle and electrochemical measurements. Selective electron transfer towards positively and negatively charged redox species was recorded. While the presence of Fe(CN){sub 6}{sup 4-} could be detected on the IL-modified BDD interface, no surface-immobilized Ru(NH{sub 3}){sub 6}{sup 3+} was recorded. The IL-modified BDD electrode showed in addition changes in surface wettability when immersed into aqueous solution containing different anions.

Influence of the covalent immobilization of graphene oxide in poly(vinyl alcohol) on human osteoblast response.

Science.gov (United States)

Linares, Javier; Matesanz, María Concepción; Feito, María José; Salavagione, Horacio Javier; Martínez, Gerardo; Gómez-Fatou, Marián; Portolés, María Teresa

2016-02-01

The differences in the response of human Saos-2 osteoblasts to nanocomposites of poly(vinyl alcohol) (PVA) and 1.5wt.% graphene oxide (GO) prepared by covalent linking (PVA/GO-c) and simple blending (PVA/GO-m) have been evaluated through different biocompatibility parameters. The effects produced on osteoblasts by these two nanocomposites were analysed in parallel and compared with the direct action of GO and with the effect of PVA films without GO. The intracellular content of reactive oxygen species (ROS) and the levels of interleukin-6 (IL-6) were measured to evaluate oxidative stress induction and protective response, respectively. The results demonstrate that the combination of GO with PVA reduces both the proliferation delay and the internal cell complexity alterations induced by GO on human osteoblasts. Moreover, the covalent attachment of GO to the PVA chains increases both cell viability and IL-6 levels, reducing both apoptosis and intracellular ROS content when compared to simple blending of both materials. The use of this strategy to modulate the biointerface reduces the toxic effects of graphene while preserving the reinforcement characteristics for application in tissue engineering scaffolds, and has enormous interest for polymer/graphene biomaterials development. Copyright © 2015 Elsevier B.V. All rights reserved.

Preparation and characterization of magnetic levan particles as matrix for trypsin immobilization

Energy Technology Data Exchange (ETDEWEB)

Maciel, J.C. [Programa de Pos-Graduacao em Ciencias Biologicas, Universidade Federal de Pernambuco, Cidade Universitaria, 50670-901 Recife, PE (Brazil); Andrad, P.L. [Programa de Pos-Graduacao em Ciencia de Materiais, Universidade Federal de Pernambuco, Cidade Universitaria, 50679-901 Recife, PE (Brazil); Neri, D.F.M., E-mail: davidfmneri@yahoo.com.br [Universidade Federal do Vale do Sao Francisco, 56304-205 Petrolina, PE (Brazil); Carvalho, L.B. [Departamento de Bioquimica, Universidade Federal de Pernambuco, Cidade Universitaria, 50679-901 Recife, PE (Brazil); Cardoso, C.A. [Departamento de Fisica, Universidade Federal de Sao Carlos, 13565-905 Sao Carlos, PE (Brazil); Calazans, G.M.T. [Departamento de Antibioticos, Universidade Federal de Pernambuco, Cidade Universitaria, 50670-901 Recife, PE (Brazil); Albino Aguiar, J. [Departamento de Fisica, Universidade Federal de Pernambuco, Cidade Universitaria, 50679-901 Recife, PE (Brazil); Silva, M.P.C. [Departamento de Bioquimica, Universidade Federal de Pernambuco, Cidade Universitaria, 50679-901 Recife, PE (Brazil)

2012-04-15

Magnetic levan was synthesized by co-precipitating D-fructofuranosyl homopolysaccharide with a solution containing Fe{sup 2+} and Fe{sup 3+} in alkaline conditions at 100 Degree-Sign C. The magnetic levan particles were characterized by scanning electron microscopy (SEM), magnetization measurements, X-ray diffractometry (XRD) and infrared spectroscopy (IR). Afterwards, magnetic levan particles were functionalized by NaIO{sub 4} oxidation and used as matrices for trypsin covalent immobilization. Magnetite and magnetic levan particles were both heterogeneous in shape and levan-magnetite presented bigger sizes compared to magnetite according to SEM images. Magnetic levan particles exhibited a magnetization 10 times lower as compared to magnetite ones, probably, due to the coating layer. XRD diffractogram showed that magnetite is the dominant phase in the magnetic levan. Infrared spectroscopy showed characteristics absorption bands of levan and magnetite (O-H, C-O-C and Fe-O bonds). The immobilized trypsin derivative was reused 10 times and lost 16% of its initial specific activity only. Therefore, these magnetic levan particles can be proposed as an alternative matrices for enzyme immobilization. - Highlights: Black-Right-Pointing-Pointer The magnetic levan particles presented larger size variation than magnetite particles due to the changes produced by coating. Black-Right-Pointing-Pointer The utilization of magnetic levan particles showed to be efficacious for immobilization of enzymes as trypsin. Black-Right-Pointing-Pointer Magnetic particles can be planned as other matrix for immobilization of biomolecule in various division processes in biotechnology.

Co-immobilization of cyclohexanone monooxygenase and glucose-6-phosphate dehydrogenase onto polyethylenimine-porous agarose polymeric composite using γ irradiation to use in biotechnological processes

International Nuclear Information System (INIS)

Atia, K.S.

2005-01-01

The co-immobilization of cyclohexanone monooxygenase (CHMO) and glucose-6-phosphate dehydrogenase (G6PDH) was optimized by completely coating, via covalent immobilization, the surface aldehyde groups of porous agarose (glyoxyl-agarose) with amine groups of polyethylenimine (PEI). The highest immobilization efficiency (∼87%) (activity of enzyme per amount of immobilized enzyme) was obtained with a CHMO/G6PDH ratio 2:1. The effects of different ratios of the support to the amount of enzymes (CHMO:G6PDH=2:1), the optimum incubation pH and the incubation time on the enzymatic activity of the enzymes were determined and found to be 5:1, 8.5 and 30 min, respectively. Subjecting the co-immobilized enzymes to doses of γ-radiation (5-100 kGy) resulted in complete loss in the activity of the free enzymes at a dose of 40 kGy, while the co-immobilized ones showed relatively high resistance to γ-radiation up to a dose of 50 kGy

Highly stable and reusable immobilized formate dehydrogenases: Promising biocatalysts for in situ regeneration of NADH

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Barış Binay

2016-02-01

Full Text Available This study aimed to prepare robust immobilized formate dehydrogenase (FDH preparations which can be used as effective biocatalysts along with functional oxidoreductases, in which in situ regeneration of NADH is required. For this purpose, Candida methylica FDH was covalently immobilized onto Immobead 150 support (FDHI150, Immobead 150 support modified with ethylenediamine and then activated with glutaraldehyde (FDHIGLU, and Immobead 150 support functionalized with aldehyde groups (FDHIALD. The highest immobilization yield and activity yield were obtained as 90% and 132%, respectively when Immobead 150 functionalized with aldehyde groups was used as support. The half-life times (t1/2 of free FDH, FDHI150, FDHIGLU and FDHIALD were calculated as 10.6, 28.9, 22.4 and 38.5 h, respectively at 35 °C. FDHI150, FDHIGLU and FDHIALD retained 69, 38 and 51% of their initial activities, respectively after 10 reuses. The results show that the FDHI150, FDHIGLU and FDHIALD offer feasible potentials for in situ regeneration of NADH.

Horseradish Peroxidase (HRP Immobilized Poly(aniline-co-m-aminophenol Film Electrodes–fabrication and Evaluation as Hydrogen Peroxide Sensor

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Seong-Ho Choi

2007-05-01

Full Text Available Enzyme modified electrodes were fabricated with poly(aniline-co-m-aminophenol. Electrochemical polymerization of aniline and m-aminophenol wasperformed to get the film of copolymer on the surface of gold electrode. Modifiedelectrodes were fabricated by two methods, physical entrapment and covalent cross-linking.In one of the method, gold nanoparticles were loaded into the copolymer film andhorseradish peroxidase (HRP was immobilized into the Au nanoparticle loaded copolymerfilm through physical entrapment. In the other method, the amino and -OH groups in thecopolymer are utilized to form covalent functionalization with HRP via glutaric dialdehydeas cross-linker/mediator. The conducting copolymer/enzyme modified electrodes preparedby physical entrapment/covalent functionalization of enzyme were tested forelectrocatalytic activities towards sensing of H2O2. Amperometric results indicate thatenzyme modified electrode via physical entrapment possesses better electrocatalyticperformance over covalent functionalized enzyme electrode.

Levan-type fructooligosaccharide production using Bacillus licheniformis RN-01 levansucrase Y246S immobilized on chitosan beads

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Surawut Sangmanee

2016-06-01

Full Text Available Bacillus licheniformis RN-01 levansucrase Y246S (LsRN-Y246S was immobilized by covalently linking onto chitosan, Sepabead EC-EP, and Sepabead EC-HFA, beads. The stability of immobilized LsRN-Y246S was found to be the highest with chitosan beads, retaining more than 70% activity after 13 weeks storage at 4 oC, and 68% activity after 12 hours incubation at 40°C. LsRN-Y246S immobilized on chitosan beads withstands sucrose concentrations up to 70% (w/v, retaining over 85% of its activity, significantly better than LsRN-Y246S immobilized on others supporting matrices. LsRN-Y246S immobilized on chitosan showed a 2.4 fold increase in activity in the presence of Mn2+, and gave slight protection against deactivation by of Cu2+, Zn2+, Fe3+, SDS and EDTA. A maximum of 8.36 g and an average of 7.35 g LFOS yield at least up to DP 11 can be produced from 25 g of sucrose, during five production cycles. We have demonstrated that LFOS can be effectively produced by chitosan immobilized LsRN-Y246S and purified.

Galactose oxidase immobilized on silica in an analytical determination of galactose-containing carbohydrates.

Science.gov (United States)

Kondakova, Lyudmila; Yanishpolskii, Victor; Tertykh, Valentin; Buglova, Tat'yana

2007-01-01

Galactose oxidase from Fusarium graminearum IMV-1060 adsorbed on, and covalently bound to, silica carriers has been used for analytical determinations of D-galactose and galactose-containing sugars. Using a flowing oxygen electrode of the Clark-type, sensor system for enzymatic analysis of water solutions of galactose-containing carbohydrates was made. Measurements were taken both in the pulse and continuous modes of a substrate flowing through a column with an immobilized biocatalyst. The linear measurement ranges for galactose-containing carbohydrates concentrations were determined.

From Protein Engineering to Immobilization: Promising Strategies for the Upgrade of Industrial Enzymes

Science.gov (United States)

Singh, Raushan Kumar; Tiwari, Manish Kumar; Singh, Ranjitha; Lee, Jung-Kul

2013-01-01

Enzymes found in nature have been exploited in industry due to their inherent catalytic properties in complex chemical processes under mild experimental and environmental conditions. The desired industrial goal is often difficult to achieve using the native form of the enzyme. Recent developments in protein engineering have revolutionized the development of commercially available enzymes into better industrial catalysts. Protein engineering aims at modifying the sequence of a protein, and hence its structure, to create enzymes with improved functional properties such as stability, specific activity, inhibition by reaction products, and selectivity towards non-natural substrates. Soluble enzymes are often immobilized onto solid insoluble supports to be reused in continuous processes and to facilitate the economical recovery of the enzyme after the reaction without any significant loss to its biochemical properties. Immobilization confers considerable stability towards temperature variations and organic solvents. Multipoint and multisubunit covalent attachments of enzymes on appropriately functionalized supports via linkers provide rigidity to the immobilized enzyme structure, ultimately resulting in improved enzyme stability. Protein engineering and immobilization techniques are sequential and compatible approaches for the improvement of enzyme properties. The present review highlights and summarizes various studies that have aimed to improve the biochemical properties of industrially significant enzymes. PMID:23306150

From protein engineering to immobilization: promising strategies for the upgrade of industrial enzymes.

Science.gov (United States)

Singh, Raushan Kumar; Tiwari, Manish Kumar; Singh, Ranjitha; Lee, Jung-Kul

2013-01-10

Enzymes found in nature have been exploited in industry due to their inherent catalytic properties in complex chemical processes under mild experimental and environmental conditions. The desired industrial goal is often difficult to achieve using the native form of the enzyme. Recent developments in protein engineering have revolutionized the development of commercially available enzymes into better industrial catalysts. Protein engineering aims at modifying the sequence of a protein, and hence its structure, to create enzymes with improved functional properties such as stability, specific activity, inhibition by reaction products, and selectivity towards non-natural substrates. Soluble enzymes are often immobilized onto solid insoluble supports to be reused in continuous processes and to facilitate the economical recovery of the enzyme after the reaction without any significant loss to its biochemical properties. Immobilization confers considerable stability towards temperature variations and organic solvents. Multipoint and multisubunit covalent attachments of enzymes on appropriately functionalized supports via linkers provide rigidity to the immobilized enzyme structure, ultimately resulting in improved enzyme stability. Protein engineering and immobilization techniques are sequential and compatible approaches for the improvement of enzyme properties. The present review highlights and summarizes various studies that have aimed to improve the biochemical properties of industrially significant enzymes.

Immobilization-stabilization of proteins on nanofibrillated cellulose derivatives and their bioactive film formation.

Science.gov (United States)

Arola, Suvi; Tammelin, Tekla; Setälä, Harri; Tullila, Antti; Linder, Markus B

2012-03-12

In a number of different applications for enzymes and specific binding proteins a key technology is the immobilization of these proteins to different types of supports. In this work we describe a concept for protein immobilization that is based on nanofibrillated cellulose (NFC). NFC is a form of cellulose where fibers have been disintegrated into fibrils that are only a few nanometers in diameter and have a very large aspect ratio. Proteins were conjugated through three different strategies using amine, epoxy, and carboxylic acid functionalized NFC. The conjugation chemistries were chosen according to the reactive groups on the NFC derivatives; epoxy amination, heterobifunctional modification of amino groups, and EDC/s-NHS activation of carboxylic acid groups. The conjugation reactions were performed in solution and immobilization was performed by spin coating the protein-NCF conjugates. The structure of NFC was shown to be advantageous for both protein performance and stability. The use of NFC allows all covalent chemistry to be performed in solution, while the immobilization is achieved by a simple spin coating or spreading of the protein-NFC conjugates on a support. This allows more scalable methods and better control of conditions compared to the traditional methods that depend on surface reactions.

Electron population uncertainty and atomic covalency

International Nuclear Information System (INIS)

Chesnut, D.B.

2006-01-01

The atoms-in-molecules (AIM) index of atomic covalency is directly related to the AIM atomic population uncertainty. The covalent bond order, delocalization index, and, therefore, the atomic covalency are maximal when electron pairs are equally shared by the atoms involved. When polarization effects are present, these measures of covalent bond character decrease. We present atomic covalences for the single- and double-heavy atom hydrides of elements of the first and second low rows of the periodic table to illustrate these effects. Some usual behavior is seen in hydrogen-bridged species due in some cases to stronger than expected multicenter bonds and in other cases to many atoms contributing to the covalency index

Site-Directed Immobilization of BMP-2: Two Approaches for the Production of Innovative Osteoinductive Scaffolds.

Science.gov (United States)

Tabisz, Barbara; Schmitz, Werner; Schmitz, Michael; Luehmann, Tessa; Heusler, Eva; Rybak, Jens-Christoph; Meinel, Lorenz; Fiebig, Juliane E; Mueller, Thomas D; Nickel, Joachim

2017-03-13

The regenerative potential of bone is strongly impaired in pathological conditions, such as nonunion fractures. To support bone regeneration various scaffolds have been developed in the past, which have been functionalized with osteogenic growth factors such as bone morphogenetic proteins (BMPs). However, most of them required supra-physiological levels of these proteins leading to burst releases, thereby causing severe side effects. Site-specific, covalent coupling of BMP2 to implant materials might be an optimal strategy in order to overcome these problems. Therefore, we created a BMP-2 variant (BMP2-K3Plk) containing a noncanonical amino acid (propargyl-l-lysine) substitution introduced by genetic code expansion that allows for site-specific and covalent immobilization onto polymeric scaffold materials. To directly compare different coupling strategies, we also produced a BMP2 variant containing an additional cysteine residue (BMP2-A2C) allowing covalent coupling by thioether formation. The BMP2-K3Plk mutant was coupled to functionalized beads by a copper-catalyzed azide-alkyne cycloaddition (CuAAC) either directly or via a short biotin-PEG linker both with high specificity. After exposing the BMP-coated beads to C2C12 cells, ALP expression appeared locally restricted in close proximity to these beads, showing that both coupled BMP2 variants trigger cell differentiation. The advantage of our approach over non-site-directed immobilization techniques is the ability to produce fully defined osteogenic surfaces, allowing for lower BMP2 loads and concomitant higher bioactivities, for example, due to controlled orientation toward BMP2 receptors. Such products might provide superior bone healing capabilities with potential safety advantages as of homogeneous product outcome.

Synthesis and effect of modification on methacylate - acrylate microspheres for Trametes versicolor laccase enzyme immobilization

Science.gov (United States)

Mazlan, Siti Zulaikha; Hanifah, Sharina Abu

2014-09-01

Immobilization of laccase on the modified copolymer methacrylate-acrylate microspheres was studied. A poly (glycidyl methacrylate-co-n-butyl acrylate) microsphere consists of epoxy groups were synthesized using suspension photocuring technique. The epoxy group in poly (GMA-nBA) microspheres were converted into amino groups with aldehyde group. Laccase immobilization is based on having the amino groups on the enzyme surface and aldehyde group on the microspheres via covalent binding. Fourier transform infrared spectroscopy (FT-IR) analysis proved the successful surface modification on microspheres. The FTIR spectrum shows the characteristic peaks at 1646 cm-1 assigned to the conformation of the polymerization that took place between monomer GMA and nBA respectively. In addition, after modification, FTIR peaks that assigned to the epoxy ring (844 cm-1 and 904 cm-1) were decreased. The results obtained from FTIR method signify good agreement with the epoxy content method. Hence, the activity of the laccase-immobilized microspheres increased upon increasing the epoxy content. Furthermore, poly (GMA-nBA) exhibited uniform microspheres with below 2 μm surface. Immobilized enzyme showed a broader pH profile and higher temperature compared native enzyme.

Effects of Temperature and pH on Immobilized Laccase Activity in Conjugated Methacrylate-Acrylate Microspheres

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Siti Zulaikha Mazlan

2017-01-01

Full Text Available Immobilization of laccase on the functionalized methacrylate-acrylate copolymer microspheres was studied. Poly(glycidyl methacrylate-co-n-butyl acrylate microspheres consisting of epoxy groups were synthesized using facile emulsion photocuring technique. The epoxy groups in poly(GMA-co-nBA microspheres were then converted to amino groups. Laccase immobilization is based on covalent binding via amino groups on the enzyme surface and aldehyde group on the microspheres. The FTIR spectra showed peak at 1646 cm−1 assigned to the conformation of the polymerization that referred to GMA and nBA monomers, respectively. After modification of the polymer, intensity of FTIR peaks assigned to the epoxy ring at 844 cm−1 and 904 cm−1 was decreased. The results obtained from FTIR exhibit a good agreement with the epoxy content method. The activity of laccase-immobilized microspheres increased upon increasing the epoxy content. Furthermore, poly(GMA-co-nBA microspheres revealed uniform size below 2 µm that contributes to large surface area of the microspheres to be used as a matrix, thus increasing the enzyme capacity and enzymatic reaction. Immobilized enzyme also shifted to higher pH and temperature compared to free enzyme.

Hydrolysis of whey lactose by immobilized β-galactosidase in a bioreactor with a spirally wound membrane.

Science.gov (United States)

Vasileva, Nastya; Ivanov, Yavor; Damyanova, Stanka; Kostova, Iliana; Godjevargova, Tzonka

2016-01-01

The β-galactosidase was covalently immobilized onto a modified polypropylene membrane, using glutaraldehyde. The optimal conditions for hydrolysis of lactose (4.7%) by immobilized β-galactosidase in a batch process were determined 13.6 U enzyme activity, 40°C, pH 6.8 and 10h. The obtained degree of hydrolysis was compared with results received by a free enzyme. It was found, that the lactose hydrolysis by an immobilized enzyme was 1.6 times more effective than the lactose hydrolysis by a free enzyme. It was determined that the stability of the immobilized enzyme was 2 times higher in comparison with the stability of free enzyme. The obtained immobilized system β-galactosidase/polypropylene membrane was applied to produce glucose-galactose syrup from waste whey. The whey characteristics and the different preliminary treatments of the whey were investigated. Then the whey lactose hydrolysis in a bioreactor by an immobilized enzyme on a spirally wound membrane was performed. The optimal membrane surface and the optimal flow rate of the whey through the membrane module were determined, respectively 100 cm(2) and 1.0 mL min(-1). After 10h, the degree of lactose hydrolysis was increased to 91%. The operation stability was studied. After 20th cycle the yield of bioreactor was 69.7%. Copyright © 2015 Elsevier B.V. All rights reserved.

Design of epoxy-functionalized Fe3O4@MCM-41 core-shell nanoparticles for enzyme immobilization.

Science.gov (United States)

Ulu, Ahmet; Ozcan, Imren; Koytepe, Suleyman; Ates, Burhan

2018-05-01

The scope of our research was to prepare the organosilane-modified Fe 3 O 4 @MCM-41 core-shell magnetic nanoparticles, used for L-ASNase immobilization and explored screening of immobilization conditions such as pH, temperature, thermal stability, kinetic parameters, reusability and storage stability. In this content, Fe 3 O 4 core-shell magnetic nanoparticles were prepared via co-precipitation method and coated with MCM-41. Then, Fe 3 O 4 @MCM-41 magnetic nanoparticles were functionalized by (3-glycidyloxypropyl) trimethoxysilane (GPTMS) as an organosilane compound. Subsequently, L-ASNase was covalently immobilized on epoxy-functionalized Fe 3 O 4 @MCM-41 magnetic nanoparticles. The immobilized L-ASNase had greater activity at high pH and temperature values. It also maintained >92% of the initial activity after incubation at 55 °C for 3 h. Regarding kinetic values, immobilized L-ASNase showed a higher Vmax and lower Km compared to native L-ASNase. In addition, it displayed excellent reusability for 12 successive cycles. After 30 days of storage at 4 °C and 25 °C, immobilized L-ASNase retained 54% and 26% of its initial activities while native L-ASNase lost about 68% and 84% of its initial activity, respectively. As a result, the immobilization of L-ASNase onto magnetic nanoparticles may provide an advantage in terms of removal of L-ASNase from reaction media. Copyright © 2018. Published by Elsevier B.V.

Artificial Intelligence Techniques to Optimize the EDC/NHS-Mediated Immobilization of Cellulase on Eudragit L-100

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Min-Chao He

2012-06-01

Full Text Available Two artificial intelligence techniques, namely artificial neural network (ANN and genetic algorithm (GA were combined to be used as a tool for optimizing the covalent immobilization of cellulase on a smart polymer, Eudragit L-100. 1-Ethyl-3-(3-dimethyllaminopropyl carbodiimide (EDC concentration, N-hydroxysuccinimide (NHS concentration and coupling time were taken as independent variables, and immobilization efficiency was taken as the response. The data of the central composite design were used to train ANN by back-propagation algorithm, and the result showed that the trained ANN fitted the data accurately (correlation coefficient R² = 0.99. Then a maximum immobilization efficiency of 88.76% was searched by genetic algorithm at a EDC concentration of 0.44%, NHS concentration of 0.37% and a coupling time of 2.22 h, where the experimental value was 87.97 ± 6.45%. The application of ANN based optimization by GA is quite successful.

Useful oriented immobilization of antibodies on chimeric magnetic particles: direct correlation of biomacromolecule orientation with biological activity by AFM studies.

Science.gov (United States)

Marciello, Marzia; Filice, Marco; Olea, David; Velez, Marisela; Guisan, José M; Mateo, Cesar

2014-12-16

The preparation and performance of a suitable chimeric biosensor based on antibodies (Abs) immobilized on lipase-coated magnetic particles by means of a standing orienting strategy are presented. This novel system is based on hydrophobic magnetic particles coated with modified lipase molecules able to orient and further immobilize different Abs in a covalent way without any previous site-selective chemical modification of biomacromolecules. Different key parameters attending the process were studied and optimized. The optimal preparation was performed using a controlled loading (1 nmol Ab g(-1) chimeric support) at pH 9 and a short reaction time to recover a biological activity of about 80%. AFM microscopy was used to study and confirm the Abs-oriented immobilization on lipase-coated magnetic particles and the final achievement of a highly active and recyclable chimeric immune sensor. This direct technique was demonstrated to be a powerful alternative to the indirect immunoactivity assay methods for the study of biomacromolecule-oriented immobilizations.

Development of Cellulose Nano fibre (CNF) Derived From Kenaf Bast Fibre and Its Potential in Enzyme Immobilization Support

International Nuclear Information System (INIS)

Safwan Sulaiman; Mohd Noriznan Mokhtar; Mohd Nazli Naim; Azhari Samsu Baharuddin

2016-01-01

This research mainly focuses on developing a natural cellulose nano fibre (CNF) from kenaf bast fibre and its potential for enzyme immobilization support. CNF was isolated by using a combination between chemical and mechanical treatments such as alkaline process and high-intensity ultrasonication process to increase the efficiency of hemicelluloses and lignin removal, and to reduce its size into nano-order. The morphological study was carried out by using scanning electron microscope (SEM), indicating most of CNF diameter in range of 50-90 nm was obtained. The result of chemical analysis shows that cellulose content of raw bast fibre, bleached pulp fibre and CNF are 66.4 %, 83.7 % and 90.0 %, respectively. By decreasing the size of cellulose fibre, it increases the number of (O-H) group on the surface that plays as important role in enzyme immobilization. Covalent immobilization of cyclodextrin glucanotransferase (CGTase) onto CNF support resulted in about 95.0 % of protein loading with 69.48 % of enzyme activity, indicating high immobilization yield of enzyme. The enzymatic reaction of immobilized CGTase was able to produce more than 40 % yield of α-CD. Reusability profile of immobilized CGTase resulted in more than 60 % of retained activity up to 7 cycles. Therefore, the CNF is highly potential to be applied as enzyme immobilization support. (author)

A novel method to immobilize collagen on polypropylene film as substrate for hepatocyte culture

International Nuclear Information System (INIS)

Zhang Yong; Wang Wenjie; Feng Qingling; Cui Fuzhai; Xu Yingxin

2006-01-01

To improve the biocompatibility of polypropylene membrane with hepatocytes, film was firstly allowed to generate peroxide groups by ammonium peroxydisulfate (APS) thermal decomposition and was then carboxylated by free radical grafting polymerization using acrylic acid as the monomer and ferrous(II) ions as redox initiator. Collagen was then covalently immobilized through amide bonds between the residue amine groups and carboxylic ones in the presence of water-soluble carbodiimide. The film was characterized by Fourier transformed infrared spectroscopy in attenuated total reflection mode (ATR-FTIR), X-photoelectron spectroscopy (XPS) and confocal laser scanning microscopy (CLSM), etc. The experimental results show that there exist hydroperoxide groups after APS aqueous thermal decomposition, which are subsequently converted into the polymeric free radicals initiating the vinyl monomers to subject grafting polymerization. The maximum collagen immobilization content is about 10.5 μg/cm 2 by ninhydrin assay. Hepatocytes cultured on collagen immobilized film show stable phenotype and normal liver-specific functions more than 20 days as observed by scanning electronic microscopy (SEM) and biochemical parameters determination

A novel method to immobilize collagen on polypropylene film as substrate for hepatocyte culture

Energy Technology Data Exchange (ETDEWEB)

Zhang Yong [Biomaterials Laboratory, Department of Material Science and Engineering, Tsinghua University, Beijing 100084 (China); Wang Wenjie [Biomaterials Laboratory, Department of Material Science and Engineering, Tsinghua University, Beijing 100084 (China); Feng Qingling [Biomaterials Laboratory, Department of Material Science and Engineering, Tsinghua University, Beijing 100084 (China)]. E-mail: biomater@mail.tsinghua.edu.cn; Cui Fuzhai [Biomaterials Laboratory, Department of Material Science and Engineering, Tsinghua University, Beijing 100084 (China); Xu Yingxin [Institute of General Surgery, Chinese PLA General Hospital, Beijing 100853 (China)

2006-05-15

To improve the biocompatibility of polypropylene membrane with hepatocytes, film was firstly allowed to generate peroxide groups by ammonium peroxydisulfate (APS) thermal decomposition and was then carboxylated by free radical grafting polymerization using acrylic acid as the monomer and ferrous(II) ions as redox initiator. Collagen was then covalently immobilized through amide bonds between the residue amine groups and carboxylic ones in the presence of water-soluble carbodiimide. The film was characterized by Fourier transformed infrared spectroscopy in attenuated total reflection mode (ATR-FTIR), X-photoelectron spectroscopy (XPS) and confocal laser scanning microscopy (CLSM), etc. The experimental results show that there exist hydroperoxide groups after APS aqueous thermal decomposition, which are subsequently converted into the polymeric free radicals initiating the vinyl monomers to subject grafting polymerization. The maximum collagen immobilization content is about 10.5 {mu}g/cm{sup 2} by ninhydrin assay. Hepatocytes cultured on collagen immobilized film show stable phenotype and normal liver-specific functions more than 20 days as observed by scanning electronic microscopy (SEM) and biochemical parameters determination.

Live cell imaging compatible immobilization of Chlamydomonas reinhardtii in microfluidic platform for biodiesel research.

Science.gov (United States)

Park, Jae Woo; Na, Sang Cheol; Nguyen, Thanh Qua; Paik, Sang-Min; Kang, Myeongwoo; Hong, Daewha; Choi, Insung S; Lee, Jae-Hyeok; Jeon, Noo Li

2015-03-01

This paper describes a novel surface immobilization method for live-cell imaging of Chlamydomonas reinhardtii for continuous monitoring of lipid droplet accumulation. Microfluidics allows high-throughput manipulation and analysis of single cells in precisely controlled microenvironment. Fluorescence imaging based quantitative measurement of lipid droplet accumulation in microalgae had been difficult due to their intrinsic motile behavior. We present a simple surface immobilization method using gelatin coating as the "biological glue." We take advantage of hydroxyproline (Hyp)-based non-covalent interaction between gelatin and the outer cell wall of microalgae to anchor the cells inside the microfluidic device. We have continuously monitored single microalgal cells for up to 6 days. The immobilized microalgae remain viable (viability was comparable to bulk suspension cultured controls). When exposed to wall shear stress, most of the cells remain attached up to 0.1 dyne/cm(2) . Surface immobilization allowed high-resolution, live-cell imaging of mitotic process in real time-which followed previously reported stages in mitosis of suspension cultured cells. Use of gelatin coated microfluidics devices can result in better methods for microalgae strain screening and culture condition optimization that will help microalgal biodiesel become more economically viable. © 2014 Wiley Periodicals, Inc.

Covalent attachment of cell-adhesive peptide Gly-Arg-Gly-Asp (GRGD) to poly(etheretherketone) surface by tailored silanization layers technique

International Nuclear Information System (INIS)

Zheng, Yanyan; Xiong, Chengdong; Li, Xiaoyu; Zhang, Lifang

2014-01-01

Highlights: • The carbonyl groups on PEEK surface were effectively reduced to hydroxyl groups using sodium borohydride. • Silanization layers technique was employed to immobilize the cell-adhesive peptide Gly-Arg-Gly-Asp (GRGD) on hydroxylation-pretreated PEEK sheet surface by covalent chemical attachment. • XPS, surface profiler and water contact angle measurements proved the presence of GRGD on PEEK surface. • Osteoblast-like cells (MC3T3-E1) attachment and proliferation were improved effectively on GRGD-modified PEEK surface. - Abstract: Poly(etheretherketone) (PEEK) is a rigid semicrystalline polymer that combines excellent mechanical properties, broad chemical resistance and bone-like stiffness and is widely used in biomedical fields. However, PEEK is naturally bioinert, leading to limited biomedical applications, especially when a direct bone-implant osteointegration is desired. In this study, a three-step reaction procedure was employed to immobilize the cell-adhesive peptide Gly-Arg-Gly-Asp (GRGD) on the surface of PEEK sheet by covalent chemical attachment to favor cell adhesion and proliferation. First, hydroxylation-pretreated PEEK surfaces were silanized with 7-Oct-1-enyltrichlorosilane (OETS) in dry cyclohexane, resulting in a silanization layer with terminal ethenyl. Second, the terminal ethylenic double bonds of the silanization layer on PEEK surface were converted to carboxyl groups through acidic potassium manganate oxidation. Finally, GRGD was covalently attached by carbodiimide mediated condensation between the carboxyl on PEEK surface and amine presents in GRGD. X-ray photoelectron spectroscopy (XPS), attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy, surface profiler and water contact angle measurements were applied to characterize the modified surfaces. The effect of cells attachment and proliferation on each specimen was investigated. Pre-osteoblast cells (MC3T3-E1) attachment, spreading and proliferation

Covalent attachment of cell-adhesive peptide Gly-Arg-Gly-Asp (GRGD) to poly(etheretherketone) surface by tailored silanization layers technique

Energy Technology Data Exchange (ETDEWEB)

Zheng, Yanyan [Chengdu Institute of Organic Chemistry, Chinese Academy of Sciences, Chengdu 610041 (China); University of Chinese Academy of Sciences, Beijing 100049 (China); Xiong, Chengdong [Chengdu Institute of Organic Chemistry, Chinese Academy of Sciences, Chengdu 610041 (China); Li, Xiaoyu [State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu 610041 (China); Zhang, Lifang, E-mail: zhanglfcioc@163.com [Chengdu Institute of Organic Chemistry, Chinese Academy of Sciences, Chengdu 610041 (China)

2014-11-30

Highlights: • The carbonyl groups on PEEK surface were effectively reduced to hydroxyl groups using sodium borohydride. • Silanization layers technique was employed to immobilize the cell-adhesive peptide Gly-Arg-Gly-Asp (GRGD) on hydroxylation-pretreated PEEK sheet surface by covalent chemical attachment. • XPS, surface profiler and water contact angle measurements proved the presence of GRGD on PEEK surface. • Osteoblast-like cells (MC3T3-E1) attachment and proliferation were improved effectively on GRGD-modified PEEK surface. - Abstract: Poly(etheretherketone) (PEEK) is a rigid semicrystalline polymer that combines excellent mechanical properties, broad chemical resistance and bone-like stiffness and is widely used in biomedical fields. However, PEEK is naturally bioinert, leading to limited biomedical applications, especially when a direct bone-implant osteointegration is desired. In this study, a three-step reaction procedure was employed to immobilize the cell-adhesive peptide Gly-Arg-Gly-Asp (GRGD) on the surface of PEEK sheet by covalent chemical attachment to favor cell adhesion and proliferation. First, hydroxylation-pretreated PEEK surfaces were silanized with 7-Oct-1-enyltrichlorosilane (OETS) in dry cyclohexane, resulting in a silanization layer with terminal ethenyl. Second, the terminal ethylenic double bonds of the silanization layer on PEEK surface were converted to carboxyl groups through acidic potassium manganate oxidation. Finally, GRGD was covalently attached by carbodiimide mediated condensation between the carboxyl on PEEK surface and amine presents in GRGD. X-ray photoelectron spectroscopy (XPS), attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy, surface profiler and water contact angle measurements were applied to characterize the modified surfaces. The effect of cells attachment and proliferation on each specimen was investigated. Pre-osteoblast cells (MC3T3-E1) attachment, spreading and proliferation

Immobilization of epidermal growth factor on titanium and stainless steel surfaces via dopamine treatment

Energy Technology Data Exchange (ETDEWEB)

Kang, Jeonghwa [Nano Medical Engineering Laboratory, RIKEN Advanced Science Institute, 2-1 Hirosawa, Wako, Saitama, 351-0198 (Japan); Department of Biological Sciences, Tokyo Metropolitan University, 1-1 Minami-Osawa, Tokyo, 192-0397 Japan (Japan); Sakuragi, Makoto; Shibata, Aya; Abe, Hiroshi; Kitajima, Takashi; Tada, Seiichi [Nano Medical Engineering Laboratory, RIKEN Advanced Science Institute, 2-1 Hirosawa, Wako, Saitama, 351-0198 (Japan); Mizutani, Masayoshi; Ohmori, Hitoshi [Material Fabrication Laboratory, RIKEN Advanced Science Institute, 2-1 Hirosawa, Wako, Saitama, 351-0198 (Japan); Ayame, Hirohito [Diagnostic Biochip Laboratory, RIKEN Center for Intellectual Property Strategies, 2-1 Hirosawa, Wako, Saitama, 351-0198 (Japan); Son, Tae Il [Bioscience and Biotechnology, Chung-Ang University, 40-1 San, Nae-Ri, Daeduck-myun, Ansung-si, Kyungki-do, 456-756 (Korea, Republic of); Aigaki, Toshiro [Department of Biological Sciences, Tokyo Metropolitan University, 1-1 Minami-Osawa, Tokyo, 192-0397 Japan (Japan); Ito, Yoshihiro, E-mail: y-ito@riken.jp [Nano Medical Engineering Laboratory, RIKEN Advanced Science Institute, 2-1 Hirosawa, Wako, Saitama, 351-0198 (Japan); Department of Biological Sciences, Tokyo Metropolitan University, 1-1 Minami-Osawa, Tokyo, 192-0397 Japan (Japan); Diagnostic Biochip Laboratory, RIKEN Center for Intellectual Property Strategies, 2-1 Hirosawa, Wako, Saitama, 351-0198 (Japan)

2012-12-01

Titanium and stainless steel were modified with dopamine for the immobilization of biomolecules, epidermal growth factor (EGF). First, the treatment of metal surfaces with a dopamine solution under different pH conditions was investigated. At higher pH, the dopamine solution turned brown and formed precipitates. Treatment of the metals with dopamine at pH 8.5 also resulted in the development of brown color at the surface of the metals. The hydrophobicity of the surfaces increased after treatment with dopamine, independently of pH. X-ray photoelectron spectroscopy revealed the formation of a significant amount of an organic layer on both surfaces at pH 8.5. According to ellipsometry measurements, the organic layer formed at pH 8.5 was about 1000 times as thick as that formed at pH 4.5. The amount of amino groups in the layer formed at pH 8.5 was also higher than that observed in the layer formed at pH 4.5. EGF molecules were immobilized onto the dopamine-treated surfaces via a coupling reaction using carbodiimide. A greater amount of EGF was immobilized on surfaces treated at pH 8.5 compared with pH 4.5. Significantly higher growth of rat fibroblast cells was observed on the two EGF-immobilized surfaces compared with non-immobilized surfaces in the presence of EGF. The present study demonstrated that metals can become bioactive via the surface immobilization of a growth factor and that the effect of the immobilized growth factor on metals was greater than that of soluble growth factor. - Highlights: Black-Right-Pointing-Pointer Epidermal growth factor was covalently immobilized on titan or stainless steel surfaces. Black-Right-Pointing-Pointer Amino groups were formed on the surfaces by the treatment and the growth factor was immobilized through amide bonds. Black-Right-Pointing-Pointer The immobilized epidermal growth factor accelerated cell proliferation more than soluble ones on the surfaces.

Lipase from Aspergillus niger obtained from mangaba residue fermentation: biochemical characterization of free and immobilized enzymes on a sol-gel matrix

Directory of Open Access Journals (Sweden)

Elis Augusta Leite dos Santos

2017-02-01

Full Text Available In this study, mangaba residue (seeds was used as a substrate for Aspergillus niger lipase production by solid-state fermentation. The partially purified enzyme was efficiently immobilized in a sol-gel matrix by covalent bonding with an immobilization yield of 91.2%. The immobilized biocatalyst and free lipase had an optimum pH of 2.0 and 5.0, respectively. However, greater stability was obtained at pH 4.0 and 7.0, respectively. The biocatalysts showed stability at the optimum temperature of 55°C, where the residual activity was above 87% after 240 min., of incubation. The lower deactivation constant (kd and higher half-life of the immobilized biocatalyst indicated greater thermal stability than those obtained with the free enzyme. The Michaelis Constant (Km (77 and 115 mM for free and immobilized lipase, respectively and maximum reaction rate (Vmax (1250 and 714 U mg-1 for free and immobilized lipase, respectively indicated that the immobilization process reduced enzyme-substrate affinity. Regarding the operational stability, the biocatalyst showed relative activity above 50% until seven cycles of reuse in olive oil hydrolysis. This novel biocatalyst obtained from a tropical fruit residue showed biochemical characteristics that support its application in future biocatalysis studies.

Design of a papain immobilized antimicrobial food package with curcumin as a crosslinker.

Directory of Open Access Journals (Sweden)

Cynthya Maria Manohar

Full Text Available Contamination of food products by spoilage and pathogenic microorganisms during post process handling is one of the major causes for food spoilage and food borne illnesses. The present green sustainable approach describes the covalent immobilization of papain to LDPE (low density polyethylene, HDPE (high density polyethylene, LLDPE (linear low density polyethylene and PCL (polycaprolactam with curcumin as the photocrosslinker. About 50% of curcumin and 82-92% of papain were successfully immobilized on these polymers. After 30 days, the free enzyme retained 87% of its original activity, while the immobilized enzyme retained more than 90% of its activity on these polymers. Papain crosslinked to LLDPE exhibited the best antibiofilm properties against Acinetobacter sp. KC119137.1 and Staphylococcus aureus NCIM 5021 when compared to the other three polymers, because of the highest amount of enzyme immobilized on this surface. Papain acts by damaging the cell membrane. The enzyme is able to reduce the amount of carbohydrate and protein contents in the biofilms formed by these organisms. Meat wrapped with the modified LDPE and stored at 4°C showed 9 log reduction of these organisms at the end of the seventh day when compared to samples wrapped with the bare polymer. This method of crosslinking can be used on polymers with or without functional groups and can be adopted to bind any type of antimicrobial agent.

Design of a Papain Immobilized Antimicrobial Food Package with Curcumin as a Crosslinker

Science.gov (United States)

Sivakumar, Ponnurengam Malliappan; Doble, Mukesh

2015-01-01

Contamination of food products by spoilage and pathogenic microorganisms during post process handling is one of the major causes for food spoilage and food borne illnesses. The present green sustainable approach describes the covalent immobilization of papain to LDPE (low density polyethylene), HDPE (high density polyethylene), LLDPE (linear low density polyethylene) and PCL (polycaprolactam) with curcumin as the photocrosslinker. About 50% of curcumin and 82-92% of papain were successfully immobilized on these polymers. After 30 days, the free enzyme retained 87% of its original activity, while the immobilized enzyme retained more than 90% of its activity on these polymers. Papain crosslinked to LLDPE exhibited the best antibiofilm properties against Acinetobacter sp. KC119137.1 and Staphylococcus aureus NCIM 5021 when compared to the other three polymers, because of the highest amount of enzyme immobilized on this surface. Papain acts by damaging the cell membrane. The enzyme is able to reduce the amount of carbohydrate and protein contents in the biofilms formed by these organisms. Meat wrapped with the modified LDPE and stored at 4°C showed 9 log reduction of these organisms at the end of the seventh day when compared to samples wrapped with the bare polymer. This method of crosslinking can be used on polymers with or without functional groups and can be adopted to bind any type of antimicrobial agent. PMID:25906061

Design of a papain immobilized antimicrobial food package with curcumin as a crosslinker.

Science.gov (United States)

Manohar, Cynthya Maria; Prabhawathi, Veluchamy; Sivakumar, Ponnurengam Malliappan; Doble, Mukesh

2015-01-01

Contamination of food products by spoilage and pathogenic microorganisms during post process handling is one of the major causes for food spoilage and food borne illnesses. The present green sustainable approach describes the covalent immobilization of papain to LDPE (low density polyethylene), HDPE (high density polyethylene), LLDPE (linear low density polyethylene) and PCL (polycaprolactam) with curcumin as the photocrosslinker. About 50% of curcumin and 82-92% of papain were successfully immobilized on these polymers. After 30 days, the free enzyme retained 87% of its original activity, while the immobilized enzyme retained more than 90% of its activity on these polymers. Papain crosslinked to LLDPE exhibited the best antibiofilm properties against Acinetobacter sp. KC119137.1 and Staphylococcus aureus NCIM 5021 when compared to the other three polymers, because of the highest amount of enzyme immobilized on this surface. Papain acts by damaging the cell membrane. The enzyme is able to reduce the amount of carbohydrate and protein contents in the biofilms formed by these organisms. Meat wrapped with the modified LDPE and stored at 4°C showed 9 log reduction of these organisms at the end of the seventh day when compared to samples wrapped with the bare polymer. This method of crosslinking can be used on polymers with or without functional groups and can be adopted to bind any type of antimicrobial agent.

Comparative performance of microbial lipases immobilized on magnetic polysiloxane polyvinyl alcohol particles

Directory of Open Access Journals (Sweden)

Laura Maria Bruno

2008-10-01

Full Text Available Microbial lipase from Mucor miehei and Candida rugosa were immobilized by covalent binding onto magnetized polysiloxane polyvinyl alcohol particles (POS-PVA. The resulting immobilized derivatives were evaluated in aqueous solution (olive oil hydrolysis and organic solvent (butyl butyrate synthesis. Higher catalytic activities were found when the coupling procedure was made with M. miehei lipase. Immobilized M. miehei lipase also showed a better operational stability and a higher half-life than C. rugosa lipase after the successive batches of esterification. The performance of C. rugosa immobilized derivative was constrained by the low lipase loading used in the immobilizing step. Further information regarding the both immobilized derivatives was also obtained through chemical composition (FTIR.Lipases microbianas de Mucor miehei e Candida rugosa foram imobilizadas por ligação covalente em partículas magnetizadas de polisiloxano-álcool polivinílico (POS-PVA. Os derivados imobilizados resultantes foram avaliados em solução aquosa (hidrólise de azeite de oliva e em solvente orgânico (síntese de butirato de butila. As maiores atividades catalíticas foram encontradas quando o procedimento de ligação foi realizado com lipase de M. miehei. O derivado imobilizado de lipase de M. miehei também apresentou melhores resultados de estabilidade operacional e tempo de meia-vida do que o de lipase de C. rugosa, após sucessivas bateladas de esterificação. O desempenho do derivado imobilizado de C. rugosa foi restringido pelo baixo carregamento de lipase usado na etapa de imobilização. Informações adicionais a respeito de ambos derivados imobilizados também foram obtidas através da composição química (FTIR.

Immobilization of acetylcholinesterase via biocompatible interface of silk fibroin for detection of organophosphate and carbamate pesticides

Energy Technology Data Exchange (ETDEWEB)

Xue Rui [College of Environmental and Energy Engineering, Beijing University of Technology, Beijing 100124 (China); Kang Tianfang, E-mail: kangtf@yahoo.cn [College of Environmental and Energy Engineering, Beijing University of Technology, Beijing 100124 (China); Lu Liping; Cheng Shuiyuan [College of Environmental and Energy Engineering, Beijing University of Technology, Beijing 100124 (China)

2012-06-01

An amperometric biosensor for the detection of organophosphate and carbamate pesticides was developed based on the immobilization of acetylcholinesterase (AChE) on regenerated silk fibroin (SF) matrix by non-covalent adsorption. SF and AChE were coated sequentially on the surface of the glassy carbon electrode (GCE) which was modified with multiwall carbon nanotube (MWNTs). The obtained biosensor was denoted as AChE-SF/MWNTs/GCE. The atomic force microscopy images showed that the SF matrix provided a more homogeneous interface for the AChE immobilization. The aggregation of immobilizing AChE was therefore avoided. The cyclic voltammogram of thiocholine at this biosensor exhibited a well defined oxidation peak at 0.667 V (vs. SCE). The inhibition rate of methyl parathion to the immobilized AChE was proportional to the logarithm of the concentration of methyl parathion over the range of the concentration of methyl parathion from 3.5 Multiplication-Sign 10{sup -6} to 2.0 Multiplication-Sign 10{sup -3} M with a detection limit of 5.0 Multiplication-Sign 10{sup -7} M. Similarly, the linearly response range of carbaryl was from 1.0 Multiplication-Sign 10{sup -7} to 3.0 Multiplication-Sign 10{sup -5} M with a detection limit of 6.0 Multiplication-Sign 10{sup -8} M. The experimental results indicate that AChE not only can be immobilized steadily on the SF matrix, but also the bioactivity of immobilizing AChE can be preserved effectively.

Effect of C/N Ratio and Media Optimization through Response Surface Methodology on Simultaneous Productions of Intra- and Extracellular Inulinase and Invertase from Aspergillus niger ATCC 20611

Science.gov (United States)

Dinarvand, Mojdeh; Rezaee, Malahat; Masomian, Malihe; Jazayeri, Seyed Davoud; Zareian, Mohsen; Abbasi, Sahar; Ariff, Arbakariya B.

2013-01-01

The study is to identify the extraction of intracellular inulinase (exo- and endoinulinase) and invertase as well as optimization medium composition for maximum productions of intra- and extracellular enzymes from Aspergillus niger ATCC 20611. From two different methods for extraction of intracellular enzymes, ultrasonic method was found more effective. Response surface methodology (RSM) with a five-variable and three-level central composite design (CCD) was employed to optimize the medium composition. The effect of five main reaction parameters including sucrose, yeast extract, NaNO3, Zn+2, and Triton X-100 on the production of enzymes was analyzed. A modified quadratic model was fitted to the data with a coefficient of determination (R 2) more than 0.90 for all responses. The intra-extracellular inulinase and invertase productions increased in the range from 16 to 8.4 times in the optimized medium (10% (w/v) sucrose, 2.5% (w/v) yeast extract, 2% (w/v) NaNO3, 1.5 mM (v/v) Zn+2, and 1% (v/v) Triton X-100) by RSM and from around 1.2 to 1.3 times greater than in the medium optimized by one-factor-at-a-time, respectively. The results of bioprocesses optimization can be useful in the scale-up fermentation and food industry. PMID:24151605

Effect of C/N Ratio and Media Optimization through Response Surface Methodology on Simultaneous Productions of Intra- and Extracellular Inulinase and Invertase from Aspergillus niger ATCC 20611

Directory of Open Access Journals (Sweden)

Mojdeh Dinarvand

2013-01-01

Full Text Available The study is to identify the extraction of intracellular inulinase (exo- and endoinulinase and invertase as well as optimization medium composition for maximum productions of intra- and extracellular enzymes from Aspergillus niger ATCC 20611. From two different methods for extraction of intracellular enzymes, ultrasonic method was found more effective. Response surface methodology (RSM with a five-variable and three-level central composite design (CCD was employed to optimize the medium composition. The effect of five main reaction parameters including sucrose, yeast extract, NaNO3, Zn+2, and Triton X-100 on the production of enzymes was analyzed. A modified quadratic model was fitted to the data with a coefficient of determination (R2 more than 0.90 for all responses. The intra-extracellular inulinase and invertase productions increased in the range from 16 to 8.4 times in the optimized medium (10% (w/v sucrose, 2.5% (w/v yeast extract, 2% (w/v NaNO3, 1.5 mM (v/v Zn+2, and 1% (v/v Triton X-100 by RSM and from around 1.2 to 1.3 times greater than in the medium optimized by one-factor-at-a-time, respectively. The results of bioprocesses optimization can be useful in the scale-up fermentation and food industry.

Immobilization of β-galactosidase from Kluyveromyces lactis onto polymeric membrane surfaces: effect of surface characteristics.

Science.gov (United States)

Güleç, Hacı Ali

2013-04-01

The aim of this study was to investigate the effects of surface characteristics of plain and plasma modified cellulose acetate (CA) membranes on the immobilization yield of β-galactosidases from Kluyveromyces lactis (KLG) and its galacto-oligosaccharide (GOS) yield, respectively. Low pressure plasma treatments involving oxygen plasma activation, plasma polymerization (PlsP) of ethylenediamine (EDA) and PlsP of 2-mercaptoethanol were used to modify plain CA membrane surfaces. KLG enzyme was immobilized onto plain and oxygen plasma treated membrane surfaces by simple adsorption. Oxygen plasma activation increased the hydrophylicity of CA membrane surfaces and it improved the immobilization yield of the enzyme by 42%. KLG enzyme was also immobilized onto CA membrane surfaces through amino groups created by PlsP of EDA via covalent binding. Plasma action at 60W plasma power and 15 min. exposure time improved the amount of membrane bounded enzyme by 3.5-fold. The enrichment of the amount of amino groups via polyethyleneimine (PEI) addition enhanced this increase from 3.5-fold to 4.5-fold. Although high enzyme loading was achived (65-83%), both of the methods dramatically decreased the enzyme activity (11-12%) and GOS yield due to probably negative effects of active amino groups. KLG enzyme was more effectively immobilized onto thiolated CA membrane surface created by PlsP of 2-mercaptoethanol with high immobilization yield (70%) and especially high enzyme activity (46%). Immobilized enzymes on the CA membranes treated by PlsP were successively reutilized for 5-8 cycles at 25°C and enzymatic derivatives retained approximately 75-80% of their initial activites at the end of the reactions. Copyright © 2012 Elsevier B.V. All rights reserved.

Sponges with covalently tethered amines for high-efficiency carbon capture

KAUST Repository

Qi, Genggeng

2014-12-12

© 2014 Macmillan Publishers Limited. All rights reserved. Adsorption using solid amine sorbents is an attractive emerging technology for energy-efficient carbon capture. Current syntheses for solid amine sorbents mainly based on physical impregnation or grafting-to methods (for example, aminosilane-grafting) lead to limited sorbent performance in terms of stability and working capacity, respectively. Here we report a family of solid amine sorbents using a grafting-from synthesis approach and synthesized by cationic polymerization of oxazolines on mesoporous silica. The sorbent with high amount of covalently tethered amines shows fast adsorption rate, high amine efficiency and sorbent capacity well exceeding the highest value reported to date for lowerature carbon dioxide sorbents under simulated flue gas conditions. The demonstrated efficiency of the new amine-immobilization chemistry may open up new avenues in the development of advanced carbon dioxide sorbents, as well as other nitrogen-functionalized systems.

Covalent assembly of poly(ethyleneimine) via layer-by-layer deposition for enhancing surface density of protein and bacteria attachment

Energy Technology Data Exchange (ETDEWEB)

Xia, Bing, E-mail: xiabing@njfu.edu.cn [Key Laboratory of Forest Genetics and Biotechnology (Ministry of Education of China), Nanjing Forestry University, Nanjing 210037 (China); Advanced Analysis and Testing Center, Nanjing Forestry University, Nanjing 210037 (China); Shi, Jisen; Dong, Chen; Zhang, Wenyi; Lu, Ye [Key Laboratory of Forest Genetics and Biotechnology (Ministry of Education of China), Nanjing Forestry University, Nanjing 210037 (China); Guo, Ping [Nanjing College of Information Technology, Nanjing 210023 (China)

2014-02-15

Covalently assembly of low molecular weight poly(ethyleneimine) was introduced to glass surfaces via glutaraldehyde crosslinking, with focus on its application on protein immobilization or bacteria attachment. Characterizations of Fourier transform infrared spectroscopy and ellipsometry measurement revealed a stepwise growth of poly(ethyleneimine) films by layer-by-layer deposition. After fluorescein isothiocyanate labelling, photoluminescence spectroscopy measurement indicated that the amount of surface accessible amine groups had been gradually enhanced with increasing poly(ethyleneimine) layers deposition. As compared with traditional aminosilanized surfaces, the surface density of amine groups was enhanced by ∼11 times after five layers grafting, which resulted in ∼9-time increasing of surface density of immobilized bovine serum albumin. Finally, these as-prepared PEI multi-films with excellent biocompatibility were adopted as culture substrates to improve Escherichia coli adherence, which showed that their surface density had been increased by ∼251 times.

Surface functionalization of cyclic olefin copolymer with aryldiazonium salts: A covalent grafting method

International Nuclear Information System (INIS)

Brisset, Florian; Vieillard, Julien; Berton, Benjamin; Morin-Grognet, Sandrine; Duclairoir-Poc, Cécile; Le Derf, Franck

2015-01-01

Graphical abstract: - Highlights: • An effective method to modify cyclic olefin copolymer surface. • The surface of COC was modified by covalent grafting of aryl diazonium salts. • The wettability of COC surface was modulated by diazonium salts. • Photoinitiation and chemical reduction have to be combined to graft diazonium salt on COC surface. - Abstract: Covalent immobilization of biomolecules on the surface of cyclic olefin copolymer (COC) is still a tough challenge. We developed a robust method for COC surface grafting through reaction with aryldiazonium. Chemical diazonium reduction generated an aryl radical and the formation of a grafted film layer on the organic surface. We also demonstrated that the chemical reduction of diazonium salt was not sufficient to form a film on the COC surface. UV illumination had to be combined with chemical reduction to graft an aryl layer onto the COC surface. We optimized organic film deposition by using different chemical reducers, different reaction times and reagent proportions. We characterized surface modifications by fluorescence microscopy and contact angle measurements, infrared spectroscopy, X-ray photoemission spectroscopy and Raman spectroscopy, and assessed the topography of the aryl film by atomic force microscopy. This original strategy allowed us to evidence various organic functions to graft biomolecules onto COC surfaces with a fast and efficient technique

Surface functionalization of cyclic olefin copolymer with aryldiazonium salts: A covalent grafting method

Energy Technology Data Exchange (ETDEWEB)

Brisset, Florian, E-mail: florian.brisset@etu.univ-rouen.fr [UMR CNRS 6014 COBRA, FR 3038, Université de Rouen, 55 rue Saint Germain, 27000 Evreux (France); Vieillard, Julien, E-mail: julien.vieillard@univ-rouen.fr [UMR CNRS 6014 COBRA, FR 3038, Université de Rouen, 55 rue Saint Germain, 27000 Evreux (France); Berton, Benjamin, E-mail: benjamin.berton@univ-rouen.fr [EA 3233 SMS, Université de Rouen, 1 rue du 7ème Chasseurs, BP281, 27002 Evreux Cedex (France); Morin-Grognet, Sandrine, E-mail: sandrine.morin@univ-rouen.fr [EA 3829 MERCI, Université de Rouen, 1 rue du 7ème Chasseurs, BP281, 27002 Evreux Cedex (France); Duclairoir-Poc, Cécile, E-mail: cecile.duclairoir@univ-rouen.fr [EA 4312 LMSM, Université de Rouen, 55 rue Saint Germain, 27000 Evreux (France); Le Derf, Franck, E-mail: franck.lederf@univ-rouen.fr [UMR CNRS 6014 COBRA, FR 3038, Université de Rouen, 55 rue Saint Germain, 27000 Evreux (France)

2015-02-28

Graphical abstract: - Highlights: • An effective method to modify cyclic olefin copolymer surface. • The surface of COC was modified by covalent grafting of aryl diazonium salts. • The wettability of COC surface was modulated by diazonium salts. • Photoinitiation and chemical reduction have to be combined to graft diazonium salt on COC surface. - Abstract: Covalent immobilization of biomolecules on the surface of cyclic olefin copolymer (COC) is still a tough challenge. We developed a robust method for COC surface grafting through reaction with aryldiazonium. Chemical diazonium reduction generated an aryl radical and the formation of a grafted film layer on the organic surface. We also demonstrated that the chemical reduction of diazonium salt was not sufficient to form a film on the COC surface. UV illumination had to be combined with chemical reduction to graft an aryl layer onto the COC surface. We optimized organic film deposition by using different chemical reducers, different reaction times and reagent proportions. We characterized surface modifications by fluorescence microscopy and contact angle measurements, infrared spectroscopy, X-ray photoemission spectroscopy and Raman spectroscopy, and assessed the topography of the aryl film by atomic force microscopy. This original strategy allowed us to evidence various organic functions to graft biomolecules onto COC surfaces with a fast and efficient technique.

Enhanced saccharification of sugarcane bagasse using soluble cellulase supplemented with immobilized β-glucosidase.

Science.gov (United States)

Borges, Diogo Gontijo; Baraldo, Anderson; Farinas, Cristiane Sanchez; Giordano, Raquel de Lima Camargo; Tardioli, Paulo Waldir

2014-09-01

The β-glucosidase (BG) enzyme plays a vital role in the hydrolysis of lignocellulosic biomass. Supplementation of the hydrolysis reaction medium with BG can reduce inhibitory effects, leading to greater conversion. In addition, the inclusion of immobilized BG can be a useful way of increasing enzyme stability and recyclability. BG was adsorbed on polyacrylic resin activated by carboxyl groups (BG-PC) and covalently attached to glyoxyl-agarose (BG-GA). BG-PC exhibited similar behavior to soluble BG in the hydrolysis of cellobiose, while BG-GA hydrolyzed the same substrate at a lower rate. However, the thermal stability of BG-GA was higher than that of free BG. Hydrolysis of pretreated sugarcane bagasse catalyzed by soluble cellulase supplemented with immobilized BG improved the conversion by up to 40% after 96 h of reaction. Both derivatives remained stable up to the third cycle and losses of activity were less than 50% after five cycles. Copyright © 2014 Elsevier Ltd. All rights reserved.

Covalent immobilization of lipase onto chitosan-mesoporous silica hybrid nanomaterials by carboxyl functionalized ionic liquids as the coupling agent.

Science.gov (United States)

Xiang, Xinran; Suo, Hongbo; Xu, Chao; Hu, Yi

2018-05-01

Chitosan-mesoporous silica SBA-15 hybrid nanomaterials (CTS-SBA-15) were synthesized by means of carboxyl functionalized ionic liquids as the coupling agent. The as-prepared CTS-SBA-15 support was characterized by TEM, FTIR, TG and nitrogen adsorption-desorption techniques. Porcine pancreas lipase (PPL) was then bound to the hybrid nanomaterials by using the cross-linking reagent glutaraldehyde (GA). Further, the parameters like cross-linking concentration, time and ratio of supports to enzyme were optimized. The property of immobilized lipase were tested in detail by enzyme activity assays. The results indicated that the hybrid nanomaterials could form three-dimensional (3D) structure with homogeneous mesoporous structures and immobilized PPL revealed excellent enzymatic performance. Copyright © 2018 Elsevier B.V. All rights reserved.

Oriented antibody immobilization to polystyrene macrocarriers for immunoassay modified with hydrazide derivatives of poly(methacrylic acid

Directory of Open Access Journals (Sweden)

Vinokurova Ludmila G

2001-08-01

Full Text Available Abstract Background Hydrophobic polystyrene is the most common material for solid phase immunoassay. Proteins are immobilized on polystyrene by passive adsorption, which often causes considerable denaturation. Biological macromolecules were found to better retain their functional activity when immobilized on hydrophilic materials. Polyacrylamide is a common material for solid-phase carriers of biological macromolecules, including immunoreagents used in affinity chromatography. New macroformats for immunoassay modified with activated polyacrylamide derivatives seem to be promising. Results New polymeric matrices for immunoassay in the form of 0.63-cm balls which contain hydrazide functional groups on hydrophilic polymer spacer arms at their surface shell are synthesized by modification of aldehyde-containing polystyrene balls with hydrazide derivatives of poly(methacrylic acid. The beads contain up to 0.31 μmol/cm2 active hydrazide groups accessible for covalent reaction with periodate-oxidized antibodies. The matrices obtained allow carrying out the oriented antibody immobilization, which increases the functional activity of immunosorbents. Conclusions An efficient site-directed antibody immobilization on a macrosupport is realized. The polymer hydrophilic spacer arms are the most convenient and effective tools for oriented antibody coupling with molded materials. The suggested scheme can be used for the modification of any other solid supports containing electrophilic groups reacting with hydrazides.

Immobilization of Tyrosinase on (3-Aminopropyltriethoxysilane-Functionalized Carbon Felt-Based Flow-Through Detectors for Electrochemical Detection of Phenolic Compounds

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Zheng Zhou

2017-07-01

Full Text Available Tyrosinase (TYR was covalently immobilized onto amino-functionalized carbon felt (CF surface via glutaraldehyde (GA. Prior to the TYR-immobilization, primary amino group was introduced to the CF surface by treatment with 3-aminopropyltriethoxysilane (APTES. The resulting TYR-immobilized CF was used as a working electrode unit of an electrochemical flow-through detector for mono- and di-phenolic compounds (i.e., catechol, p-cresol, phenol and p-chlorophenol. Additionally, flow injection peaks based on electroreduction of the enzymatically produced o-quinone species were detected at −0.05 V vs. Ag/AgCl. The resulting TYR/GA/APTES/CF biosensor responded well to all compounds tested with limits of detection range from 7.5 to 35 nmol−1 (based on three times S/N ratio. Moreover, such modified electrode exhibits good stability and reproducibility for catechol. No serious degradation of the peak current was found over 30 consecutive injections.

Expression and purification of plant fructan exohydrolases and their potential applications in fructose production.

Science.gov (United States)

Zhan, Wenyue; Jin, Lijin; Jiao, Jiao; Zhang, Xi; Zhang, Yan; Zhao, Haiyan; Liang, Mingxiang

2018-03-01

Inulinases from microorganisms have been extensively studied for their role in the production of fructose from fructan. Fructan can also be hydrolyzed by plant fructan exohydrolases (FEHs), but these enzymes have not been used to produce fructose commercially. Two Ht1-FEHs (Ht1-FEH I and Ht1-FEH II) were recently characterized in Jerusalem artichoke. In this study, we cloned the third member of the Ht1-FEH family in Jerusalem artichoke (i.e., Ht1-FEH III). When heterologously expressed in Pichia pastoris X-33, Ht1-FEH III not only demonstrated hydrolysis activity towards β (2, 1)-linked fructans and β (2, 6)-linked levan, but also towards sucrose. To explore the potential industrial applications, we heterologously expressed and purified six plant 1-FEHs from two typical fructan plants (i.e., chicory and Jerusalem artichoke) and showed that chicory Ci1-FEH IIa had the highest hydrolysis capacity to fructan in vitro. Furthermore, we immobilized Ci1-FEH IIa on resin and optimized the immobilization conditions. We found that inulin-type fructan or the tuber extract from Jerusalem artichoke could be rapidly degraded into fructose and sucrose by immobilized Ci1-FEH IIa. The capacity of Ci1-FEH IIa to release fructose from fructans was comparable to that of some inulinases from microorganisms. Thus, plant FEHs have potential applications in fructose production. Copyright © 2017 Elsevier B.V. All rights reserved.

An improved amperometric L-lactate biosensor based on covalent immobilization of microbial lactate oxidase onto carboxylated multiwalled carbon nanotubes/copper nanoparticles/polyaniline modified pencil graphite electrode.

Science.gov (United States)

Dagar, Kusum; Pundir, C S

2017-01-01

An improved amperometric l-lactate biosensor was constructed based on covalent immobilization of lactate oxidase (LOx) from Pediococcus species onto carboxylated multiwalled carbon nanotubes (cMWCNT)/copper nanoparticles (CuNPs)/polyaniline (PANI) hybrid film electrodeposited on the surface of a pencil graphite electrode (PGE). The enzyme electrode was characterized by cyclic voltammetry (CV), scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy and electrochemical impedance spectroscopy (EIS), while CuNPs synthesized by chemical reduction method, were characterized by transmission electron microscopy (TEM), UV spectrascopy and X-ray diffraction (XRD). The biosensor showed maximum response within 5s at pH 8.0 in 0.05M sodium phosphate buffer and 37°C, when operated at 20mVs -1 . The biosensor had a detection limit of 0.25μM with a wide working range between 1μM-2500μM. The biosensor was employed for measurement of l-lactic acid level in plasma of apparently healthy and diseased persons. Analytical recovery of added lactic acid in plasma was 95.5%. Within- and between-batch coefficients of variations were 6.24% and 4.19% respectively. There was a good correlation (R 2 =0.97) between plasma lactate values as measured by standard enzymatic spectrophotometric method and the present biosensor. The working enzyme electrode was used 180 times over a period of 140 days, when stored at 4°C. Copyright © 2016. Published by Elsevier Inc.

Covalent attachment of proteins to solid supports and surfaces via Sortase-mediated ligation.

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Lilyan Chan

Full Text Available BACKGROUND: There is growing interest in the attachment of proteins to solid supports for the development of supported catalysts, affinity matrices, and micro devices as well as for the development of planar and bead based protein arrays for multiplexed assays of protein concentration, interactions, and activity. A critical requirement for these applications is the generation of a stable linkage between the solid support and the immobilized, but still functional, protein. METHODOLOGY: Solid supports including crosslinked polymer beads, beaded agarose, and planar glass surfaces, were modified to present an oligoglycine motif to solution. A range of proteins were ligated to the various surfaces using the Sortase A enzyme of S. aureus. Reactions were carried out in aqueous buffer conditions at room temperature for times between one and twelve hours. CONCLUSIONS: The Sortase A transpeptidase of S. aureus provides a general, robust, and gentle approach to the selective covalent immobilization of proteins on three very different solid supports. The proteins remain functional and accessible to solution. Sortase mediated ligation is therefore a straightforward methodology for the preparation of solid supported enzymes and bead based assays, as well as the modification of planar surfaces for microanalytical devices and protein arrays.

Activity behavior of a HPLC column including α-chymotrypsin immobilized monosized-porous particles

International Nuclear Information System (INIS)

Bilici, Z.; Camli, S.T.; Unsal, E.; Tuncel, A.

2004-01-01

In this study, a polymer-based, α-chymotrypsin (CT) immobilized HPLC column was prepared as a potential material for affinity-HPLC and chiral separation applications. Monosized-macroporous particles were synthesized as the support material by a relatively new polymerization protocol, the so-called, 'modified seeded polymerization'. The particles were obtained in the form of styrene-glycidyl methacrylate- divinylbenzene terpolymer approximately 11 μm in size. The particles were treated with aqueous ammonia to have primary amine groups on the porous surface. The amine functionalized particles were reacted by glutaraldehyde and the enzyme, CT, was covalently attached. CT carrying monosized-porous particles were slurry packed into the HPLC column 50 mmx4.6 mm in size. Since the activity behavior of immobilized CT played an important role in the enantiomeric separations performed by similar columns, the enzymatic activity behavior of the column produced by our protocol was determined. For this purpose, HPLC column was used as a packed bed reactor and the enzymatic reaction was continuously followed by measuring the absorbance of the output flow by the UV-detector of HPLC. S-shaped absorbance-time curves were obtained by monitoring the reactor output both in dynamic and steady-state periods. The columns with relatively lower immobilized enzyme content were more sensitive to the changes in the operating conditions and responded with more appreciable substrate conversion changes. The maximum reaction rate of the immobilized enzyme was estimated as approximately 25% of the free one by the mathematical model describing the activity behavior of the column. No significant loss was observed in the activity of the immobilized enzyme during the course of the experiments

Enzyme-Catalyzed Oxidation of 17β-Estradiol Using Immobilized Laccase from Trametes versicolor

Science.gov (United States)

Cardinal-Watkins, Chantale; Nicell, Jim A.

2011-01-01

Many natural and synthetic estrogens are amenable to oxidation through the catalytic action of oxidative enzymes such as the fungal laccase Trametes versicolor. This study focused on characterizing the conversion of estradiol (E2) using laccase that had been immobilized by covalent bonding onto silica beads contained in a bench-scale continuous-flow packed bed reactor. Conversion of E2 accomplished in the reactor declined when the temperature of the system was changed from room temperature to just above freezing at pH 5 as a result of a reduced rate of reaction rather than inactivation of the enzyme. Similarly, conversion increased when the system was brought to warmer temperatures. E2 conversion increased when the pH of the influent to the immobilized laccase reactor was changed from pH 7 to pH 5, but longer-term experiments showed that the enzyme is more stable at pH 7. Results also showed that the immobilized laccase maintained its activity when treating a constant supply of aqueous E2 at a low mean residence time over a 12-hour period and when treating a constant supply of aqueous E2 at a high mean residence time over a period of 9 days. PMID:21869925

Covalent bonding in heavy metal oxides

Energy Technology Data Exchange (ETDEWEB)

Bagus, Paul S.; Nelin, Connie J.; Hrovat, Dave A.; Ilton, Eugene S.

2017-04-07

Novel theoretical methods were used to quantify the magnitude and the energetic contributions of 4f/5f-O2p and 5d/6d-O2p interactions to covalent bonding in lanthanide and actinide oxides. Although many analyses have neglected the involvement of the frontier d orbitals, the present study shows that f and d covalency are of comparable importance. Two trends are identified. As is expected, the covalent mixing is larger when the nominal oxidation state is higher. More subtly, the importance of the nf covalent mixing decreases sharply relative to (n+1)d as the nf occupation increases. Atomic properties of the metal cations that drive these trends are identified.

Characterization of bioactive RGD peptide immobilized onto poly(acrylic acid) thin films by plasma polymerization

Energy Technology Data Exchange (ETDEWEB)

Seo, Hyun Suk; Ko, Yeong Mu; Shim, Jae Won [Department of Dental Materials, School of Dentistry, MRC Center, Chosun University, Gwangju (Korea, Republic of); Lim, Yun Kyong; Kook, Joong-Ki [Department of Oral Biochemistry, School of Dentistry, Chosun University, Gwangju (Korea, Republic of); Cho, Dong-Lyun [School of Applied Chemical Engineering and Center for Functional Nano Fine Chemicals, Chonnam National University, Gwangju (Korea, Republic of); Kim, Byung Hoon, E-mail: kim5055@chosun.ac.kr [Department of Dental Materials, School of Dentistry, MRC Center, Chosun University, Gwangju (Korea, Republic of)

2010-11-01

Plasma surface modification can be used to improve the surface properties of commercial pure Ti by creating functional groups to produce bioactive materials with different surface topography. In this study, a titanium surface was modified with acrylic acid (AA) using a plasma treatment and immobilized with bioactive arginine-glycine-aspartic acid (RGD) peptide, which may accelerate the tissue integration of bone implants. Both terminals containing the -NH{sub 2} of RGD peptide sequence and -COOH of poly(acrylic acid) (PAA) thin film were combined with a covalent bond in the presence of 1-ethyl-3-3-dimethylaminopropyl carbodiimide (EDC). The chemical structure and morphology of AA film and RGD immobilized surface were investigated by X-ray photoelectron spectroscopy (XPS), Fourier transform infrared (FT-IR), atomic force microscopy (AFM), and scanning electron microscopy (SEM). All chemical analysis showed full coverage of the Ti substrate with the PAA thin film containing COOH groups and the RGD peptide. The MC3T3-E1 cells were cultured on each specimen, and the cell alkaline phosphatase (ALP) activity were examined. The surface-immobilized RGD peptide has a significantly increased the ALP activity of MC3T3-E1 cells. These results suggest that the RGD peptide immobilization on the titanium surface has an effect on osteoblastic differentiation of MC3T3-E1 cells and potential use in osteo-conductive bone implants.

Fabrication of graphene oxide decorated with Fe3O4@SiO2 for immobilization of cellulase

Science.gov (United States)

Li, Yue; Wang, Xiang-Yu; Jiang, Xiao-Ping; Ye, Jing-Jing; Zhang, Ye-Wang; Zhang, Xiao-Yun

2015-01-01

Fe3O4@SiO2-graphene oxide (GO) composites were successfully fabricated by chemical binding of functional Fe3O4@SiO2 and GO and applied to immobilization of cellulase via covalent attachment. The prepared composites were further characterized by transmission electron microscopy and Fourier transform infrared spectroscopy. Fe3O4 nanoparticles (NPs) were monodisperse spheres with a mean diameter of 17 ± 0.2 nm. The thickness of SiO2 layer was calculated as being 6.5 ± 0.2 nm. The size of Fe3O4@SiO2 NPs was 24 ± 0.3 nm, similar to that of Fe3O4@SiO2-NH2. Fe3O4@SiO2-GO composites were synthesized by linking of Fe3O4@SiO2-NH2 NPs to GO with the catalysis of EDC and NHS. The prepared composites were used for immobilization of cellulase. A high immobilization yield and efficiency of above 90 % were obtained after the optimization. The half-life of immobilized cellulase (722 min) was 3.34-fold higher than that of free enzyme (216 min) at 50 °C. Compared with the free cellulase, the optimal temperature of the immobilized enzyme was not changed; but the optimal pH was shifted from 5.0 to 4.0, and the thermal stability was enhanced. The immobilized cellulase could be easily separated and reused under magnetic field. These results strongly indicate that the cellulase immobilized onto the Fe3O4@SiO2-GO composite has potential applications in the production of bioethanol.

Silk fibroin immobilization on poly(ethylene terephthalate) films: Comparison of two surface modification methods and their effect on mesenchymal stem cells culture

Energy Technology Data Exchange (ETDEWEB)

Liang, Meini; Yao, Jinrong; Chen, Xin; Huang, Lei; Shao, Zhengzhong, E-mail: zzshao@fudan.edu.cn

2013-04-01

Silk fibroin (SF) has played a curial role for the surface modification of conventional materials to improve the biocompatibility, and SF modified poly(ethylene terephthalate) (PET) materials have potential applications on tissue engineering such as artificial ligament, artificial vessel, artificial heart valve sewing cuffs dacron and surgical mesh engineering. In this work, SF was immobilized onto PET film via two different methods: 1) plasma pretreatment followed by SF dip coating (PET-SF) and 2) plasma-induce acrylic acid graft polymerization and subsequent covalent immobilization of SF on PET film (PET-PAA-SF). It could be found that plasma treatment provided higher surface roughness which was suitable for further SF dip coating, while grafted poly(acrylic acid) (PAA) promised the covalent bonding between SF and PAA. ATR-FTIR adsorption band at 3284 cm{sup −1}, 1623 cm{sup −1} and 1520 cm{sup −1} suggested the successful introduction of SF onto PET surface, while the amount of immobilized SF of PET-SF was higher than PET-PAA-SF according to XPS investigation (0.29 vs 0.23 for N/C ratio). Surface modified PET film was used as substrate for mesenchymal stem cells (MSCs) culture, the cells on PET-SF surface exhibited optimum density compared to PET-PAA-SF according to CCK-8 assays, which indicated that plasma pretreatment followed by SF dip coating was a simple and effective way to prepare biocompatible PET surface. Highlights: ► Silk fibroins were immobilized onto PET films with or without the linker of PAA. ► Various techniques were performed to characterize the modified surfaces ► Plasma treatment followed by SF dip coating introduced more SF onto PET films. ► Compare to PET-PAA-SF, PET-SF has better biocompatibility base on MSCs culture.

Photogeneration of singlet oxygen by the phenothiazine derivatives covalently bound to the surface-modified glassy carbon

Energy Technology Data Exchange (ETDEWEB)

Blacha-Grzechnik, Agata, E-mail: agata.blacha@polsl.pl [Faculty of Chemistry, Silesian University of Technology, Strzody 9, 44-100 Gliwice (Poland); Piwowar, Katarzyna; Krukiewicz, Katarzyna [Faculty of Chemistry, Silesian University of Technology, Strzody 9, 44-100 Gliwice (Poland); Koscielniak, Piotr; Szuber, Jacek [Institute of Electronics, Silesian University of Technology, Akademicka 16, 44-100 Gliwice (Poland); Zak, Jerzy K. [Faculty of Chemistry, Silesian University of Technology, Strzody 9, 44-100 Gliwice (Poland)

2016-05-15

Highlights: • The selected group of four NH{sub 2}-derivatives of phenothiazine was grafted to Glassy Carbon (GC) surface. • The grafted phenothiazines are able to generate {sup 1}O{sub 2} when activated by the radiation. • Such modified solid surfaces may find their application in the wastewater treatment. - Abstract: The selected group of four amine-derivatives of phenothiazine was covalently grafted to the glassy carbon surface in the four-step procedure consisting of the electrochemical reduction of the diazonium salt followed by the electrochemical and chemical post-modification steps. The proposed strategy involves the bonding of linker molecule to which the photosensitizer is attached. The synthesized organic layers were characterized by means of cyclic voltammetry, XPS and Raman Spectroscopy. It was shown that the phenothiazines immobilized via proposed strategy retain their photochemical properties and are able to generate {sup 1}O{sub 2} when activated by the laser radiation. The effectiveness of in situ singlet oxygen generation by those new solid photoactive materials was determined by means of UVVis spectroscopy. The reported, covalently modified solid surfaces may find their application as the singlet oxygen photogenerators in the fine chemicals’ synthesis or in the wastewater treatment.

Anticodeine aptamer immobilized on a Whatman cellulose paper for thin-film microextraction of codeine from urine followed by electrospray ionization ion mobility spectrometry.

Science.gov (United States)

Hashemian, Zahra; Khayamian, Taghi; Saraji, Mohammad

2015-02-01

A combination of thin-film microextaction based on an aptamer immobilized on modified Whatman cellulose paper followed by electrospray ionization ion mobility spectrometry has been developed for the analysis of codeine in urine samples. The immobilization is based on the covalent linking of an amino-modified anticodeine aptamer to aldehyde groups of the oxidized cellulose paper. The covalent bonds were examined by infrared spectroscopy and elemental analysis. The effect of the extraction parameters, including the elution conditions (solvent type and volume), extraction time, and extraction temperature, on the extraction efficiency were investigated. Under the optimized conditions, the linear dynamic range was found to be 10-300 ng/mL with a detection limit of 3.4 ng/mL for codeine in urine. The relative standard deviation was 6.8% for three replicate measurements of codeine at 100 ng/mL in urine. Furthermore, the samples were analyzed with a standard method for the analysis of codeine using high-performance liquid chromatography with ultraviolet detection. The comparison of the results validates the accuracy of the proposed method as an alternative method for the analysis of codeine in urine samples.

Binding of alpha-fetoprotein by immobilized monoclonal antibodies during episodes of zero-gravity obtained by parabolic flight

Science.gov (United States)

Spooner, Brian S.; Guikema, James A.; Barnes, Grady

1990-01-01

Alpha-fetoprotein (AFP), a single-chain polypeptide which is synthesized by the liver and yolk sac of the human fetus, provided a model ligand for assessing the effects of microgravity on ligand binding to surface-immobilized model receptor molecules. Monoclonal antibodies, used as receptors for AFP, were immobilized by covalent attachment to latex microparticles. Zero gravity environment was obtained by parabolic flight aboard NASA 930, a modified KC-135 aircraft. Buring the onset of an episode of zero gravity, ligand and receptor were mixed. Timed incubation (20 s) was terminated by centrifugation, the supernatant removed, and microparticies were assessed for bound AFP by immunochemical methods. The extent of binding was not influenced by microgravity, when compared with 1-G controls, which suggests that aberrant cellular activities observed in microgravity are not the simple expression of altered macromolecular interactions.

Immobilization of catalase on poly(acrylonitrile)-g.co-hydroxyethyl methacrylate

International Nuclear Information System (INIS)

Cavaco, M.C.; Andrade, M.E.

1991-01-01

Various poly(acrylonitrile)-g.co-hydroxyethyl methacrylate graft copolymers were prepared by using gamma irradiation at 400 Gy.h -1 . The influence of monomer concentration and time of irradiation on the level of grafting were analysed. The hydrophilicity of the polymeric supports was calculated by determining the water sorption. From the results obtained, we could conclude that the hydrophilicity was dependent on the yield of grafting. Some of the graft copolymers prepared were used for the immobilization of catalase. This enzyme was covalently coupled to the hydroxyl groups of the support after activation either with epichlorohydrin or with p-toluene sulphonyl chloride. The yield of enzyme coupling increases when hexamethylenediamine was used as a 'spacer'. (author) 5 refs.; 3 figs.; 2 tabs

Magnetic Fe3O4@MCM-41 core-shell nanoparticles functionalized with thiol silane for efficient l-asparaginase immobilization.

Science.gov (United States)

Ulu, Ahmet; Noma, Samir Abbas Ali; Koytepe, Suleyman; Ates, Burhan

2018-06-06

l-Asparaginase (l-ASNase) is a vital enzyme for medical treatment and food industry. Here, we assessed the use of Fe 3 O 4 @Mobil Composition of Matter No. 41 (MCM-41) magnetic nanoparticles as carrier matrix for l-ASNase immobilization. In addition, surface of Fe 3 O 4 @MCM-41 magnetic nanoparticles was functionalized with 3-mercaptopropyltrimethoxysilane (MPTMS) to enhance stability of l-ASNase. The chemical structure, thermal properties, magnetic profile and morphology of the thiol-functionalized Fe 3 O 4 @MCM-41 magnetic nanoparticles were characterized with Fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), differential thermal analysis (DTA), differential scanning calorimetry (DSC), vibrating sample magnetometer (VSM), scanning electron microscope (SEM), energy dispersive X-ray (EDX) spectroscopy and zeta-potential measurement. l-ASNase was covalently immobilized onto the thiol-functionalized Fe 3 O 4 @MCM-41 magnetic nanoparticles. The properties of the immobilized enzyme, including optimum pH, temperature, kinetic parameters, thermal stability, reusability and storage stability were investigated and compared to free one. Immobilized enzyme was found to be stable over a wide range of pH and temperature range than free enzyme. The immobilized l-ASNase also showed higher thermal stability after 180 min incubation at 50 °C. The immobilized enzyme still retained 63% of its original activity after 16 times of reuse. The Km value for the immobilized enzyme was 1.15-fold lower than the free enzyme, which indicates increased affinity for the substrate. Additionally, the immobilized enzyme was active over 65% and 53% after 30 days of storage at 4 °C and room temperature (∼25 °C), respectively. Thereby, the results confirmed that thiol-functionalized Fe 3 O 4 @MCM-41 magnetic nanoparticles had high efficiency for l-ASNase immobilization and improved stability of L-ASNase.

Increased Protein Structural Resolution from Diethylpyrocarbonate-based Covalent Labeling and Mass Spectrometric Detection

Science.gov (United States)

Zhou, Yuping; Vachet, Richard W.

2012-04-01

Covalent labeling and mass spectrometry are seeing increased use together as a way to obtain insight into the 3-dimensional structure of proteins and protein complexes. Several amino acid specific (e.g., diethylpyrocarbonate) and non-specific (e.g., hydroxyl radicals) labeling reagents are available for this purpose. Diethylpyrocarbonate (DEPC) is a promising labeling reagent because it can potentially probe up to 30% of the residues in the average protein and gives only one reaction product, thereby facilitating mass spectrometric analysis. It was recently reported, though, that DEPC modifications are labile for some amino acids. Here, we show that label loss is more significant and widespread than previously thought, especially for Ser, Thr, Tyr, and His residues, when relatively long protein digestion times are used. Such label loss ultimately decreases the amount of protein structural information that is obtainable with this reagent. We find, however, that the number of DEPC modified residues and, thus, protein structural information, can be significantly increased by decreasing the time between the covalent labeling reaction and the mass spectrometric analysis. This is most effectively accomplished using short (e.g., 2 h) proteolytic digestions with enzymes such as immobilized chymotrypsin or Glu-C rather than using methods (e.g., microwave or ultrasonic irradiation) that accelerate proteolysis in other ways. Using short digestion times, we show that the percentage of solvent accessible residues that can be modified by DEPC increases from 44% to 67% for cytochrome c, 35% to 81% for myoglobin, and 76% to 95% for β-2-microglobulin. In effect, these increased numbers of modified residues improve the protein structural resolution available from this covalent labeling method. Compared with typical overnight digestion conditions, the short digestion times decrease the average distance between modified residues from 11 to 7 Å for myoglobin, 13 to 10 Å for

A Universal Protocol for Photochemical Covalent Immobilization of Intact Carbohydrates for the Preparation of Carbohydrate Microarrays

Science.gov (United States)

Wang, Huibin; Zhang, Yiming; Yuan, Xun; Chen, Yi; Yan, Mingdi

2010-01-01

A universal photochemical method has been established for the immobilization of intact carbohydrates and their analogues, and for the fabrication of carbohydrate microarrays. The method features the use of perfluorophenyl azide (PFPA)-modified substrates and the photochemical reaction of surface azido groups with printed carbohydrates. Various aldoses, ketoses, non-reducing sugars such as alditols and their derivatives can be directly arrayed on the PFPA-modified chips. The lectin-recognition ability of arrayed mannose, glucose and their oligo- and polysaccharides were confirmed using surface plasmon resonance imaging and laser-induced fluorescence imaging. PMID:21138274

Immobilization of biomolecules to plasma polymerized pentafluorophenyl methacrylate.

Science.gov (United States)

Duque, Luis; Menges, Bernhard; Borros, Salvador; Förch, Renate

2010-10-11

Thin films of plasma polymerized pentafluorophenyl methacrylate (pp-PFM) offer highly reactive ester groups throughout the structure of the film that allow for subsequent reactions with different aminated reagents and biological molecules. The present paper follows on from previous work on the plasma deposition of pentafluorophenyl methacrylate (PFM) for optimum functional group retention (Francesch, L.; Borros, S.; Knoll, W.; Foerch, R. Langmuir 2007, 23, 3927) and reactivity in aqueous solution (Duque, L.; Queralto, N.; Francesch, L.; Bumbu, G. G.; Borros, S.; Berger, R.; Förch, R. Plasma Process. Polym. 2010, accepted for publication) to investigate the binding of a biologically active peptide known to induce cellular adhesion (IKVAV) and of biochemically active proteins such as BSA and fibrinogen. Analyses of the films and of the immobilization of the biomolecules were carried out using infrared reflection absorption spectroscopy (IRRAS), X-ray photoelectron spectroscopy (XPS), and atomic force microscopy (AFM). The attachment of the biomolecules on pulsed plasma polymerized pentafluorophenyl methacrylate was monitored using surface plasmon resonance spectroscopy (SPR). SPR analysis confirmed the presence of immobilized biomolecules on the plasma polymer and was used to determine the mass coverage of the peptide and proteins adsorbed onto the films. The combined analysis of the surfaces suggests the covalent binding of the peptide and proteins to the surface of the pp-PFM.

Aspergillus niger PA2 Tyrosinase Covalently Immobilized on a Novel Eco-Friendly Bio-Composite of Chitosan-Gelatin and Its Evaluation for L-DOPA Production

Science.gov (United States)

Agarwal, Pragati; Dubey, Swati; Singh, Mukta; Singh, Rajesh P.

2016-01-01

Tyrosinase (EC 1.14.18.1) a copper-containing monooxygenase, isolated from a fungal isolate Aspergillus niger PA2 was subjected for immobilization onto a composite consisting of chitosan and gelatin biopolymers. The homogeneity of the chitosan-gelatin biocomposite film was characterized by X-ray diffraction analyses. To evaluate immobilization efficiency, chitosan-gelatin-Tyr bio-composite films were analyzed by field emission scanning electron microscopy, atomic force microscopy and UV-spectroscopy. The rough morphology of the film led to a high loading of enzyme and it could retain its bioactivity for a longer period. The enzyme adsorbed onto the film exhibited 72% of its activity after 10 days and exhibited good repeatability for up to nine times, after intermittent storage. Moreover, the immobilized enzyme exhibited broader pH and temperature profile as compared to free counterpart. Immobilized enzyme was further evaluated for the synthesis of L-DOPA (2,4-dihydroxy phenylalanine) which is a precursor of dopamine and a potent drug for the treatment of Parkinson's disease and for myocardium neurogenic injury. PMID:28066399

Plasma dye coating as straightforward and widely applicable procedure for dye immobilization on polymeric materials.

Science.gov (United States)

De Smet, Lieselot; Vancoillie, Gertjan; Minshall, Peter; Lava, Kathleen; Steyaert, Iline; Schoolaert, Ella; Van De Walle, Elke; Dubruel, Peter; De Clerck, Karen; Hoogenboom, Richard

2018-03-16

Here, we introduce a novel concept for the fabrication of colored materials with significantly reduced dye leaching through covalent immobilization of the desired dye using plasma-generated surface radicals. This plasma dye coating (PDC) procedure immobilizes a pre-adsorbed layer of a dye functionalized with a radical sensitive group on the surface through radical addition caused by a short plasma treatment. The non-specific nature of the plasma-generated surface radicals allows for a wide variety of dyes including azobenzenes and sulfonphthaleins, functionalized with radical sensitive groups to avoid significant dye degradation, to be combined with various materials including PP, PE, PA6, cellulose, and PTFE. The wide applicability, low consumption of dye, relatively short procedure time, and the possibility of continuous PDC using an atmospheric plasma reactor make this procedure economically interesting for various applications ranging from simple coloring of a material to the fabrication of chromic sensor fabrics as demonstrated by preparing a range of halochromic materials.

Radiation target analyses of free and immobilized glucose 6-phosphate dehydrogenase

International Nuclear Information System (INIS)

Kempner, E.S.; Miller, J.H.

2010-01-01

The sensitivity of the enzyme glucose 6-phosphate dehydrogenase to ionizing radiation was examined under several conditions, including the presence of several free-radical scavengers. The enzyme was also irradiated when covalently bound to polyacrylamide beads whose structure is very similar to the polypeptide backbone of proteins. All the enzyme forms were irradiated in the frozen state with high-energy electrons from a linear accelerator. Surviving enzyme activity and surviving monomers were determined; the data were analyzed by target theory. Free-radical scavengers reduced the radiation target size of both the activity and monomers of the free enzyme, but not that of the immobilized enzyme activity. The target size of the activity of the free enzyme was that of a dimer mass, but in the case of the immobilized enzyme it was equal to the smaller mass of the monomer. Free-radical scavengers reduce the target size by modifying radiation energy transfer. The target size of the polyacrylamide-bound enzyme activity was expected to be very large since the connection between polyacrylamide and protein is a peptide bond which permits transfer of radiation-deposited energy. Several explanations concerning energy transfer are suggested for this result.

Production of Biodiesel Using Immobilized Lipase and the Characterization of Different Co-Immobilizing Agents and Immobilization Methods

Directory of Open Access Journals (Sweden)

Kang Zhao

2016-08-01

Full Text Available Lipase from Candida sp. 99–125 is widely employed to catalyzed transesterification and can be used for biodiesel production. In this study, the lipase was immobilized by combined adsorption and entrapment to catalyze biodiesel production from waste cooking oil (WCO via transesterification, and investigating co-immobilizing agents as additives according to the enzyme activity. The addition of the mixed co-immobilizing agents has positive effects on the activities of the immobilized lipase. Three different immobilizing methods were compared by the conversion ratio of biodiesel and structured by Atom Force Microscopy (AFM and Scanning Electron Microscopy (SEM, respectively. It was found that entrapment followed by adsorption was the best method. The effect of the co-immobilizing agent amount, lipase dosage, water content, and reuse ability of the immobilized lipase was investigated. By comparison with previous research, this immobilized lipase showed good reuse ability: the conversion ratio excesses 70% after 10 subsequent reactions, in particular, was better than Novozym435 and TLIM on waste cooking oil for one unit of lipase.

Covalent Grafting of the RGD-Peptide onto Polyetheretherketone Surfaces via Schiff Base Formation

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Marc Becker

2013-01-01

Full Text Available In recent years, the synthetic polymer polyetheretherketone (PEEK has increasingly been used in a number of orthopedic implementations, due to its excellent mechanical properties, bioinertness, and chemical resistance. For in vivo applications, the surface of PEEK, which does not naturally support cell adhesion, has to be modified to improve tissue integration. In the present work we demonstrate a novel wet-chemical modification of PEEK to modify the surface, enabling the covalent grafting of the cell-adhesive RGD-peptide. Modification of the polymer surface was achieved via Schiff base formation using an aliphatic diamine and subsequent crosslinker-mediated immobilization of the peptide. In cell culture experiments with primary osteoblasts it was shown that the RGD-modified PEEK not only significantly promoted cellular adhesion but also strongly enhanced the proliferation of osteoblasts on the modified polymer surface.

Improvement of Aspergillus flavus saponin hydrolase thermal stability and productivity via immobilization on a novel carrier based on sugarcane bagasse

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Hala A. Amin

2018-03-01

Full Text Available Soyasapogenol B (SB is known to have many biological activities such as hepatoprotective, anti-inflammatory, anti-mutagenic, antiviral and anticancer activities. Enzymatic conversion of soyasaponins to SB was carried out using saponin hydrolase (SH extracted from Aspergillus flavus. The partially purified enzyme was immobilized on different carriers by physical adsorption, covalent binding or entrapment. Among the investigated carriers, Eupergit C and sugarcane bagasse (SCB activated by DIC and NHS were the most suitable two carriers for immobilization (the immobilized forms recovered 46.5 and 37.1% of the loaded enzyme activity, respectively. Under optimized immobilization conditions, immobilized SH on Eupergit C and on activated SBC recovered 87.7 and 83.3% of its original activity, respectively. Compared to free SH, immobilized SH on Eupergit C and on activated SCB showed higher optimum pH, activation energy, half-lives and lower deactivation constant rate. Also, their SB productivities were improved by 2.3- and 2.2-folds compared to free SH (87.7 and 83.3 vs. 37.5%, respectively. Hence, being SCB more sustainable and an inexpensive material, it can be considered a good alternative to Eupergit C as a support for SH immobilization. SH immobilization on industrially applicable and inexpensive carrier is necessary to improve SB yield and reduce its production cost. The chemical structure of SCB and the resulting cellulose derivatives were studied by ATR-IR spectroscopy. The thermal analysis technique was used to study the chemical treatment of SCB and coupling with the enzyme. This technique confirmed the removal of lignin and hemicellulose by chemical treatment of SCB.

Fabrication of graphene oxide decorated with Fe3O4@SiO2 for immobilization of cellulase

International Nuclear Information System (INIS)

Li, Yue; Wang, Xiang-Yu; Jiang, Xiao-Ping; Ye, Jing-Jing; Zhang, Ye-Wang; Zhang, Xiao-Yun

2015-01-01

Fe 3 O 4 @SiO 2 –graphene oxide (GO) composites were successfully fabricated by chemical binding of functional Fe 3 O 4 @SiO 2 and GO and applied to immobilization of cellulase via covalent attachment. The prepared composites were further characterized by transmission electron microscopy and Fourier transform infrared spectroscopy. Fe 3 O 4 nanoparticles (NPs) were monodisperse spheres with a mean diameter of 17 ± 0.2 nm. The thickness of SiO 2 layer was calculated as being 6.5 ± 0.2 nm. The size of Fe 3 O 4 @SiO 2 NPs was 24 ± 0.3 nm, similar to that of Fe 3 O 4 @SiO 2 –NH 2 . Fe 3 O 4 @SiO 2 –GO composites were synthesized by linking of Fe 3 O 4 @SiO 2 –NH 2 NPs to GO with the catalysis of EDC and NHS. The prepared composites were used for immobilization of cellulase. A high immobilization yield and efficiency of above 90 % were obtained after the optimization. The half-life of immobilized cellulase (722 min) was 3.34-fold higher than that of free enzyme (216 min) at 50 °C. Compared with the free cellulase, the optimal temperature of the immobilized enzyme was not changed; but the optimal pH was shifted from 5.0 to 4.0, and the thermal stability was enhanced. The immobilized cellulase could be easily separated and reused under magnetic field. These results strongly indicate that the cellulase immobilized onto the Fe 3 O 4 @SiO 2 –GO composite has potential applications in the production of bioethanol

Mechanical desorption of immobilized proteins using carbon dioxide aerosols for reusable biosensors

International Nuclear Information System (INIS)

Singh, Renu; Hong, Seongkyeol; Jang, Jaesung

2015-01-01

Highlights: • Immobilized proteins were removed using carbon dioxide aerosols. • We observed high removal efficiencies due to the aerosol treatment. • We confirmed the removal with FTIR and X-ray photoelectron spectroscopy. • This CO 2 aerosol treatment did not undermine re-functionalization. • This technique is a fast and damage-free method to reuse a sensor surface. - Abstract: Reusability of a biosensor has recently received considerable attention, and it is closely related with the effective desorption of probe molecules. We present a novel mechanical desorption technique to reuse biosensors by using periodic jets of carbon dioxide (CO 2 ) aerosols (a mixture of solid and gaseous CO 2 ), and demonstrate its feasibility by removing physically adsorbed and covalently bonded fluorescent proteins i.e., Escherichia coli fluorescein isothiocyanate antibody and bovine serum albumin (E. coli FITC–Ab and FITC–BSA) from silicon chips. The proteins on the chip surfaces were measured by fluorescent images before and after applying the aerosols. The removal efficiency of the aerosol treatment was measured for various concentrations (1–20 μg mL −1 ) of E. coli FITC–Ab and FITC–BSA with two different removal cycles (5 and 11 cycles; each cycle: 8 s). We observed high removal efficiencies (>93.5% for physically adsorbed Ab and >84.6% for covalently bonded Ab) at 11 cycle aerosol treatment. This CO 2 aerosol treatment did not undermine re-functionalization, which was confirmed by the fluorescent images of FITC–Abs for fresh and reused chips. Desorption of the immobilized layers was validated by Fourier transform infrared and X-ray photoelectron spectroscopic analyses. We also conducted an experiment on the regeneration of E. coli sensing chips using this aerosol treatment, and the chips were re-used 5 times successfully. This mechanical desorption technique is a highly effective and novel strategy for reusable biosensors

Covalent attachment of aptamer onto nanocomposite as a high performance electrochemical sensing platform: Fabrication of an ultra-sensitive ibuprofen electrochemical aptasensor

Energy Technology Data Exchange (ETDEWEB)

Roushani, Mahmoud, E-mail: mahmoudroushani@yahoo.com; Shahdost-fard, Faezeh

2016-11-01

In the present study, we report a selective electrochemical aptasensor for the ultrasensitive detection of an anti-inflammatory drug, ibuprofen (IBP). The proposed system was achieved by the modification of a glassy carbon electrode (GCE) with multiwalled carbon nanotubes/ionic liquid/chitosan (MWCNTs/IL/Chit) nanocomposite and the covalent immobilization of the IBP specific aptamer (Apt) onto the modified electrode surface followed by methylene blue (MB) intercalated onto the Apt as the electrochemical redox marker. Upon the incubation of the IBP as a target in the proposed aptasensor, the peak current of MB decreases due to the formation of the Apt-IBP complex and the displacement of MB from the immobilized Apt onto the modified electrode surface. The nanocomposite not only increases the electrode surface area and accelerate the electron transfer kinetics but also it provides a highly stable matrix to enhance the loading amount of the Apt DNA sequence. Through differential pulse voltammetry (DPV) experiments, it was found that the proposed aptasensor could detect the IBP with a linear range (70 pM up to 6 μM) and the detection limit (LOD) as low as 20 pM. The results showed that the aptasensor had good sensitivity, stability, reproducibility, and specificity to detect the IBP. The proposed aptasensor was successfully applied for measuring the IBP concentration in real samples. Based on our experiments we can say that the present method proposes new horizons for the development of other aptasensors for diagnostic application in biosensing. - Highlights: • An electrochemical aptasensor is developed for ultrasensitive detection of IBP. • The aptasensor is made by covalent immobilization of aptamer on a modified GCE. • A nanocomposite as a modifier provides a specific surface with high conductivity. • This nanocomposite leads to a high density of the DNA sequence on the GCE surface. • This method proposes new horizons for development other aptasensors for

Covalent attachment of aptamer onto nanocomposite as a high performance electrochemical sensing platform: Fabrication of an ultra-sensitive ibuprofen electrochemical aptasensor

International Nuclear Information System (INIS)

Roushani, Mahmoud; Shahdost-fard, Faezeh

2016-01-01

In the present study, we report a selective electrochemical aptasensor for the ultrasensitive detection of an anti-inflammatory drug, ibuprofen (IBP). The proposed system was achieved by the modification of a glassy carbon electrode (GCE) with multiwalled carbon nanotubes/ionic liquid/chitosan (MWCNTs/IL/Chit) nanocomposite and the covalent immobilization of the IBP specific aptamer (Apt) onto the modified electrode surface followed by methylene blue (MB) intercalated onto the Apt as the electrochemical redox marker. Upon the incubation of the IBP as a target in the proposed aptasensor, the peak current of MB decreases due to the formation of the Apt-IBP complex and the displacement of MB from the immobilized Apt onto the modified electrode surface. The nanocomposite not only increases the electrode surface area and accelerate the electron transfer kinetics but also it provides a highly stable matrix to enhance the loading amount of the Apt DNA sequence. Through differential pulse voltammetry (DPV) experiments, it was found that the proposed aptasensor could detect the IBP with a linear range (70 pM up to 6 μM) and the detection limit (LOD) as low as 20 pM. The results showed that the aptasensor had good sensitivity, stability, reproducibility, and specificity to detect the IBP. The proposed aptasensor was successfully applied for measuring the IBP concentration in real samples. Based on our experiments we can say that the present method proposes new horizons for the development of other aptasensors for diagnostic application in biosensing. - Highlights: • An electrochemical aptasensor is developed for ultrasensitive detection of IBP. • The aptasensor is made by covalent immobilization of aptamer on a modified GCE. • A nanocomposite as a modifier provides a specific surface with high conductivity. • This nanocomposite leads to a high density of the DNA sequence on the GCE surface. • This method proposes new horizons for development other aptasensors for

Purification and characterization of β-Fructosidase with inulinase activity from Aspergillus niger - 245

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Vinícius D'Arcadia Cruz

1998-01-01

Full Text Available Aspergillus niger - 245, a strain isolated from soil samples showed good β-fructosidase activity when inoculated in medium formulated with dahlia extract tubers. The enzyme was purified by precipitation in ammonium sulphate and percolated in DEAE-Sephadex A-50 and CM-cellulose columns, witch showed a single peack in all the purification steps, maintaining the I/S ratio between 0.32 to, 0.39. Optimum pH for inulinase activity (I was between 4.0 - 4.5 and for invertase activity (S between 2.5 and 5.0. The optimum temperature was 60O.C for both activities and no loss in activity was observed when it was maintained at this temperature for 30 min. The Km value was 1.44 and 5.0, respectively, for I and S and Vm value 10.48 and 30.55, respectively. The I activity was strongly inhibited by Hg2+ and Ag+ and 2 x 10-3 M of glucose, but not by fructose at the same concentration. The enzyme showed an exo-action mechanism, acting on the inulin of different origins. In assay conditions total hydrolysis of all the frutans was obtained, although it has shown larger activity on the chicory inulin than that one from artichoke Jerusalem and dahlia, in the first 30 min. The obtained results suggested that the enzyme presented good potential for industrial application in the preparing the fructose syrupsAspergillus niger - 245, isolado do solo mostrou boa atividade de b-frutosidase meio formulado com extrato de tubérculos de dahlia. A enzima foi purificada por precipitação em sulfato de amônia e percolada em colunas de DEAE-Sephadex A-50 e CM-celulose, produzindo um único pico em todas as fases de purificação e mantendo a relação I/S entre 0,32 a 0,39. O pH ótimo para a atividade de inulinase (I foi encontrado entre 4,0 - 4.5 e para a atividade de invertase (S em 2,5 e 5,0. A temperatura ótima foi de 60O.C para ambas as atividades e nenhuma perda foi observada quando mantida nesta temperatura por 30 min. Os valores de Km foram de 1,44 e 5

Enhanced stability and catalytic activity of immobilized α-amylase on modified Fe3O4 nanoparticles for potential application in food industries

International Nuclear Information System (INIS)

Hosseinipour, Seyyedeh Leila; Khiabani, Mahmoud Sowti; Hamishehkar, Hamed; Salehi, Roya

2015-01-01

Enzymes play an essential role in catalyzing various reactions. However, their instability upon repetitive/prolonged use, elevated temperature, acidic or alkaline pH remains an area of concern. α-Amylase, a widely used enzyme in food industries for starch hydrolysis, was covalently immobilized on the surface of two developed matrices, amino-functionalized silica-coated magnetite nanoparticles (AFSMNPs) alone and covered with chitosan. The synthesis steps and characterizations of NPs were examined by FT-IR, VSM, and SEM. Modified nanoparticles with average diameters of 20–80 nm were obtained. Enzyme immobilization efficiencies of 89 and 74 were obtained for AFSMNPs and chitosan-coated AFSMNPs, respectively. The optimum pH obtained was 6.5 and 8.0 for the enzyme immobilized on AFSMNPs and chitosan-coated AFSMNPs, respectively. Optimum temperature for the immobilized enzyme shifted toward higher temperatures. Considerable enhancements in thermal stabilities were observed for the immobilized enzyme at elevated temperatures up to 80 °C. A frequent use experiment demonstrated that the immobilized enzyme retained 74 and 85 % of its original activity even after 20 times of repeated use in AFSMNPs and chitosan-coated AFSMNPs, respectively. Storage stability demonstrated that free enzyme lost its activity completely within 30 days. But, immobilized enzyme on AFSMNPs and chitosan-coated AFSMNPs preserved 65.73 and 78.63 % of its initial activity, respectively, after 80 days of incubation. In conclusion, a substantial improvement in the performance of the immobilized enzyme with reference to the free enzyme was obtained. Furthermore, the relative activities of immobilized enzyme are superior than free enzyme over the broader pH and temperature ranges.

Enhanced stability and catalytic activity of immobilized α-amylase on modified Fe3O4 nanoparticles for potential application in food industries

Science.gov (United States)

Hosseinipour, Seyyedeh Leila; Khiabani, Mahmoud Sowti; Hamishehkar, Hamed; Salehi, Roya

2015-09-01

Enzymes play an essential role in catalyzing various reactions. However, their instability upon repetitive/prolonged use, elevated temperature, acidic or alkaline pH remains an area of concern. α-Amylase, a widely used enzyme in food industries for starch hydrolysis, was covalently immobilized on the surface of two developed matrices, amino-functionalized silica-coated magnetite nanoparticles (AFSMNPs) alone and covered with chitosan. The synthesis steps and characterizations of NPs were examined by FT-IR, VSM, and SEM. Modified nanoparticles with average diameters of 20-80 nm were obtained. Enzyme immobilization efficiencies of 89 and 74 were obtained for AFSMNPs and chitosan-coated AFSMNPs, respectively. The optimum pH obtained was 6.5 and 8.0 for the enzyme immobilized on AFSMNPs and chitosan-coated AFSMNPs, respectively. Optimum temperature for the immobilized enzyme shifted toward higher temperatures. Considerable enhancements in thermal stabilities were observed for the immobilized enzyme at elevated temperatures up to 80 °C. A frequent use experiment demonstrated that the immobilized enzyme retained 74 and 85 % of its original activity even after 20 times of repeated use in AFSMNPs and chitosan-coated AFSMNPs, respectively. Storage stability demonstrated that free enzyme lost its activity completely within 30 days. But, immobilized enzyme on AFSMNPs and chitosan-coated AFSMNPs preserved 65.73 and 78.63 % of its initial activity, respectively, after 80 days of incubation. In conclusion, a substantial improvement in the performance of the immobilized enzyme with reference to the free enzyme was obtained. Furthermore, the relative activities of immobilized enzyme are superior than free enzyme over the broader pH and temperature ranges.

Chemistry of Covalent Organic Frameworks.

Science.gov (United States)

Waller, Peter J; Gándara, Felipe; Yaghi, Omar M

2015-12-15

Linking organic molecules by covalent bonds into extended solids typically generates amorphous, disordered materials. The ability to develop strategies for obtaining crystals of such solids is of interest because it opens the way for precise control of the geometry and functionality of the extended structure, and the stereochemical orientation of its constituents. Covalent organic frameworks (COFs) are a new class of porous covalent organic structures whose backbone is composed entirely of light elements (B, C, N, O, Si) that represent a successful demonstration of how crystalline materials of covalent solids can be achieved. COFs are made by combination of organic building units covalently linked into extended structures to make crystalline materials. The attainment of crystals is done by several techniques in which a balance is struck between the thermodynamic reversibility of the linking reactions and their kinetics. This success has led to the expansion of COF materials to include organic units linked by these strong covalent bonds: B-O, C-N, B-N, and B-O-Si. Since the organic constituents of COFs, when linked, do not undergo significant change in their overall geometry, it has been possible to predict the structures of the resulting COFs, and this advantage has facilitated their characterization using powder X-ray diffraction (PXRD) techniques. It has also allowed for the synthesis of COF structures by design and for their formation with the desired composition, pore size, and aperture. In practice, the modeled PXRD pattern for a given expected COF is compared with the experimental one, and depending on the quality of the match, this is used as a starting point for solving and then refining the crystal structure of the target COF. These characteristics make COFs an attractive class of new porous materials. Accordingly, they have been used as gas storage materials for energy applications, solid supports for catalysis, and optoelectronic devices. A large and

Covalent and non-covalent functionalization and solubilization of ...

Indian Academy of Sciences (India)

Wintec

photographs of the dispersions of amide-functio- nalized DWNTs in dichloromethane and tetrahydro- furan. In figure 3b, we show a TEM image of DWNTs after covalent functionalization. The images are not as sharp after functionalization as in the case of pris- tine nanotubes (figure 3a), and the bundles seem to be intact.

Simultaneous saccharification of inulin and starch using commercial glucoamylase and the subsequent bioconversion to high titer sorbitol and gluconic acid.

Science.gov (United States)

An, Kehong; Hu, Fengxian; Bao, Jie

2013-12-01

A new bioprocess for production of sorbitol and gluconic acid from two low-cost feedstocks, inulin and cassava starch, using a commercially available enzyme was proposed in this study. The commercial glucoamylase GA-L NEW from Genencor was found to demonstrate a high inulinase activity for hydrolysis of inulin into fructose and glucose. The glucoamylase was used to replace the expensive and not commercially available inulinase enzyme for simultaneous saccharification of inulin and starch into high titer glucose and fructose hydrolysate. The glucose and fructose in the hydrolysate were converted into sorbitol and gluconic acid using immobilized whole cells of the recombinant Zymomonas mobilis strain. The high gluconic acid concentration of 193 g/L and sorbitol concentration of 180 g/L with the overall yield of 97.3 % were obtained in the batch operations. The present study provided a practical production method of sorbitol and gluconic acid from low cost feedstocks and enzymes.

Modification of silicon nitride surfaces with GOPES and APTES for antibody immobilization: computational and experimental studies

International Nuclear Information System (INIS)

To, Thien Dien; Nguyen, Anh Tuan; Phan, Khoa Nhat Thanh; Truong, An Thu Thi; Doan, Tin Chanh Duc; Dang, Chien Mau

2015-01-01

Chemical modification of silicon nitride (SiN) surfaces by silanization has been widely studied especially with 3-(aminopropyl)triethoxysilane (APTES) and 3-(glycidyloxypropyl) dimethylethoxysilane (GOPES). However few reports performed the experimental and computational studies together. In this study, surface modification of SiN surfaces with GOPES and APTES covalently bound with glutaraldehyde (GTA) was investigated for antibody immobilization. The monoclonal anti-cytokeratin-FITC (MACF) antibody was immobilized on the modified SiN surfaces. The modified surfaces were characterized by water contact angle measurements, atomic force microscopy and fluorescence microscopy. The FITC-fluorescent label indicated the existence of MACF antibody on the SiN surfaces and the efficiency of the silanization reaction. Absorption of APTES and GOPES on the oxidized SiN surfaces was computationally modeled and calculated by Materials Studio software. The computational and experimental results showed that modification of the SiN surfaces with APTES and GTA was more effective than the modification with GOPES. (paper)

The Effect of Covalently Immobilized FGF-2 on Biphasic Calcium Phosphate Bone Substitute on Enhanced Biological Compatibility and Activity

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Kyung-Suk Moon

2015-01-01

Full Text Available The purpose of this research was to covalently graft fibroblast growth factor 2 (FGF-2 onto biphasic calcium phosphate (BCP via a bifunctional cross-linker technique and to estimate the optimal dose of FGF-2 resulting in the best osteogenic differentiation of human mesenchymal stem cells (hMSCs. SEM observation revealed that the surface of the 100 ng FGF-2 coated BCP was completely covered with the nanoparticles expected to be from the silane coupling agent. XRD, FT-IR, and XPS analysis showed that silane treatment, bifunctional cross-linker coating, and FGF-2 covalent grafts were conducted successfully without deforming the crystalline structure of BCP. An MTT assay demonstrated that FGF-2 coated BCP had good biocompatibility, regardless of the concentration of FGF-2, after 24 or 48 h of incubation. An alkaline phosphatase (ALP activity assay (14 days of incubation and the ALP gene expression level of real-time PCR analysis (7 days of incubation revealed that 50, 100, and 200 ng FGF-2 coated BCP induced the highest activities among all experimental groups and control group (P<0.05. Thus, low concentrations of FGF-2 facilitated excellent osteogenesis and were effective at enhancing osteogenic potential. Also, the bifunctional cross-linker technique is expected to be a more feasible way to induce osteogenic differentiation while minimizing the risk of FGF-2 overdose.

Hydrophilic porous magnetic poly(GMA-MBAA-NVP) composite microspheres containing oxirane groups: An efficient carrier for immobilizing penicillin G acylase

Energy Technology Data Exchange (ETDEWEB)

Xue, Ping; Su, Weiguang, E-mail: weiguangsu@nxu.edu.cn; Gu, Yaohua; Liu, Haifeng; Wang, Julan

2015-03-15

Magnetic hydrophilic polymeric microspheres containing oxirane groups were prepared by inverse suspension polymerization of glycidyl methacrylate (GMA), N, N′-methylene bisacrylamide (MBAA) and N-vinyl pyrrolidone (NVP) in the existence of formamide, which were denoted as magnetic poly(GMA-MBAA-NVP) microspheres. The magnetic poly(GMA-MBAA-NVP) microspheres were characterized by scanning electron microscopy (SEM), FT-IR spectroscopy, X-ray diffraction (XRD), vibrating sample magnetometer (VSM) and so on. The results showed that poly(GMA-MBAA-NVP) microspheres possessed well spherical shape, narrow size distribution, abundant porous structure, reactive oxirane groups and superparamagnetic properties. Formamide used in the present work served as a modifier, a dispersant and a porogen to form final porous polymer microspheres. The penicillin G acylase (PGA) was covalently immobilized onto the magnetic microspheres through the reaction between the amino groups of enzyme and the oxirane groups on the microspheres for producing 6-aminopenicillanic acid (6-APA). The effects of GMA/NVP ratio and crosslink density on the activity of immobilized PGA were investigated. The highest apparent activity, enzyme loading and coupling yield of immobilized PGA were 821 IU/g, 65.3 mg/g and 42.3% respectively when the mass ratio of GMA/NVP was 1:1 and crosslink density was 60%. Compared with the free PGA, immobilized PGA showed a wider range of pH value and reaction temperature. The relative activity and reaction rate of immobilized PGA remained almost constant after 20 recycles. The magnetic poly(GMA-MBAA-NVP) microspheres would be very promising carriers for immobilizing enzymes in industrial application. - Highlights: • The magnetic poly(GMA-MBAA-NVP) microspheres were successfully synthesized. • Formamide served as a modifier, a dispersant and a porogen to form microspheres. • The magnetic microspheres were highly efficient carriers for immobilizing PGA. • Immobilized PGA

The strategies of DNA immobilization and hybridization detection mechanism in the construction of electrochemical DNA sensor: A review

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Jahwarhar Izuan Abdul Rashid

2017-11-01

Full Text Available In recent years, electrochemical deoxyribonucleic acid (DNA sensor has recently emerged as promising alternative clinical diagnostic devices especially for infectious disease by exploiting DNA recognition events and converting them into an electrochemical signal. This is because the existing DNA diagnostic method possesses certain drawbacks such as time-consuming, expensive, laborious, low selectivity and sensitivity. DNA immobilization strategies and mechanism of electrochemical detection are two the most important aspects that should be considered before developing highly selective and sensitive electrochemical DNA sensor. Here, we focus on some recent strategies for DNA probes immobilization on the surface of electrochemical transducer such as adsorption, covalent bonding and Avidin/Streptavidin-Biotin interaction on the electrode surface for specific interaction with its complementary DNA target. A numerous approach for DNA hybridization detection based electrochemical technique that frequently used including direct DNA electrochemical detection and label based electrochemical (redox-active indicator, enzyme label and nanoparticles were also discussed in aiming to provide general guide for the design of electrochemical DNA sensor. We also discussed the challenges and suggestions to improve the application of electrochemical DNA sensor at point-care setting. Keywords: Electrochemical DNA sensor, DNA immobilization, DNA hybridization, Electrochemical mechanism

Immobilization of a mediator onto carbon cloth electrode and employment of the modified electrode to an electroenzymatic bioreactor.

Science.gov (United States)

Jeong, Eun-Seon; Sathishkumar, Muthuswamy; Jayabalan, Rasu; Jeong, Su-Hyeon; Park, Song-Yie; Mun, Sung-Phil; Yun, Sei-Eok

2012-10-01

5,5'-Dithiobis(2-nitrobenzoic acid) (DTNB) was selected as an electron transfer mediator and was covalently immobilized onto high porosity carbon cloth to employ as a working electrode in an electrochemical NAD(+)-regeneration process, which was coupled to an enzymatic reaction. The voltammetric behavior of DTNB attached to carbon cloth resembled that of DTNB in buffered aqueous solution, and the electrocatalytic anodic current grew continuously upon addition of NADH at different concentrations, indicating that DTNB is immobilized to carbon cloth effectively and the immobilized DTNB is active as a soluble one. The bioelectrocatalytic NAD+ regeneration was coupled to the conversion of L-glutamate into alpha-ketoglutarate by L-glutamate dehydrogenase within the same microreactor. The conversion at 3 mM monosodium glutamate was very rapid, up to 12 h, to result in 90%, and then slow up to 24 h, showing 94%, followed by slight decrease. Low conversion was shown when substrate concentration exceeding 4 mM was tested, suggesting that L-glutamate dehydrogenase is inhibited by alpha-ketoglutarate. However, our electrochemical NAD+ regeneration procedure looks advantageous over the enzymatic procedure using NADH oxidase, from the viewpoint of reaction time to completion.

Hydrophilic porous magnetic poly(GMA-MBAA-NVP) composite microspheres containing oxirane groups: An efficient carrier for immobilizing penicillin G acylase

Science.gov (United States)

Xue, Ping; Su, Weiguang; Gu, Yaohua; Liu, Haifeng; Wang, Julan

2015-03-01

Magnetic hydrophilic polymeric microspheres containing oxirane groups were prepared by inverse suspension polymerization of glycidyl methacrylate (GMA), N, N‧-methylene bisacrylamide (MBAA) and N-vinyl pyrrolidone (NVP) in the existence of formamide, which were denoted as magnetic poly(GMA-MBAA-NVP) microspheres. The magnetic poly(GMA-MBAA-NVP) microspheres were characterized by scanning electron microscopy (SEM), FT-IR spectroscopy, X-ray diffraction (XRD), vibrating sample magnetometer (VSM) and so on. The results showed that poly(GMA-MBAA-NVP) microspheres possessed well spherical shape, narrow size distribution, abundant porous structure, reactive oxirane groups and superparamagnetic properties. Formamide used in the present work served as a modifier, a dispersant and a porogen to form final porous polymer microspheres. The penicillin G acylase (PGA) was covalently immobilized onto the magnetic microspheres through the reaction between the amino groups of enzyme and the oxirane groups on the microspheres for producing 6-aminopenicillanic acid (6-APA). The effects of GMA/NVP ratio and crosslink density on the activity of immobilized PGA were investigated. The highest apparent activity, enzyme loading and coupling yield of immobilized PGA were 821 IU/g, 65.3 mg/g and 42.3% respectively when the mass ratio of GMA/NVP was 1:1 and crosslink density was 60%. Compared with the free PGA, immobilized PGA showed a wider range of pH value and reaction temperature. The relative activity and reaction rate of immobilized PGA remained almost constant after 20 recycles. The magnetic poly(GMA-MBAA-NVP) microspheres would be very promising carriers for immobilizing enzymes in industrial application.

Plasma immersion ion implantation of polyurethane shape memory polymer: Surface properties and protein immobilization

Science.gov (United States)

Cheng, Xinying; Kondyurin, Alexey; Bao, Shisan; Bilek, Marcela M. M.; Ye, Lin

2017-09-01

Polyurethane-type shape memory polymers (SMPU) are promising biomedical implant materials due to their ability to recover to a predetermined shape from a temporary shape induced by thermal activation close to human body temperature and their advantageous mechanical properties including large recovery strains and low recovery stresses. Plasma Immersion Ion Implantation (PIII) is a surface modification process using energetic ions that generates radicals in polymer surfaces leading to carbonisation and oxidation and the ability to covalently immobilise proteins without the need for wet chemistry. Here we show that PIII treatment of SMPU significantly enhances its bioactivity making SMPU suitable for applications in permanent implantable biomedical devices. Scanning Electron Microscopy (SEM), contact angle measurements, surface energy measurements, attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy and X-ray photoelectron spectroscopy (XPS) were used to characterise the PIII modified surface, including its after treatment aging kinetics and its capability to covalently immobilise protein directly from solution. The results show a substantial improvement in wettability and dramatic changes of surface chemical composition dependent on treatment duration, due to the generation of radicals and subsequent oxidation. The SMPU surface, PIII treated for 200s, achieved a saturated level of covalently immobilized protein indicating that a full monolayer coverage was achieved. We conclude that PIII is a promising and efficient surface modification method to enhance the biocompatibility of SMPU for use in medical applications that demand bioactivity for tissue integration and stability in vivo.

Cobalt(II phthalocyanine bonded to 3-n-propylimidazole immobilized on silica gel surface: preparation and electrochemical properties

Directory of Open Access Journals (Sweden)

Fujiwara Sergio T.

1999-01-01

Full Text Available Co-Phthalocyanine complex was immobilized on 3-n-propylimidazole groups grafted on a porous SiO2 surface (specific surface area S BET = 500 m² g-1 and efficiently electrocatalyzed the oxalic acid oxidation on a carbon paste electrode surface made of this material. Intermolecular interactions of the complex species which can normally interfere in the redox process practically are not observed in the present case because of a low average surface density, delta = 4.7 x 10-13 mol cm-2 (delta = Nf/S BET, where Nf is the amount of adsorbed Co-phtalocyanine per gram of modified silica gel of the complex species material prepared. The linear response of the electrode to oxalic acid concentration, between 6.5 x 10-4 and 3.2 x 10-3 mol L-1, associated with its high chemical stability makes the covalently immobilized Co-phtalocyanine complex material very attractive in preparing a new class of chemical sensors.

Enhanced stability and catalytic activity of immobilized α-amylase on modified Fe{sub 3}O{sub 4} nanoparticles for potential application in food industries

Energy Technology Data Exchange (ETDEWEB)

Hosseinipour, Seyyedeh Leila [Tabriz University of Medical Sciences, Biotechnology Research Center (Iran, Islamic Republic of); Khiabani, Mahmoud Sowti [Tabriz University, Department of Food Science and Technology, Faculty of Agricultural Science (Iran, Islamic Republic of); Hamishehkar, Hamed, E-mail: hamishehkar.hamed@gmail.com [Tabriz University of Medical Sciences, Drug Applied Research Center (Iran, Islamic Republic of); Salehi, Roya, E-mail: salehiro@tbzmed.ac.ir [Tabriz University of Medical Sciences, Research Center for Pharmaceutical Nanotechnology and School of Advanced Medical Science (Iran, Islamic Republic of)

2015-09-15

Enzymes play an essential role in catalyzing various reactions. However, their instability upon repetitive/prolonged use, elevated temperature, acidic or alkaline pH remains an area of concern. α-Amylase, a widely used enzyme in food industries for starch hydrolysis, was covalently immobilized on the surface of two developed matrices, amino-functionalized silica-coated magnetite nanoparticles (AFSMNPs) alone and covered with chitosan. The synthesis steps and characterizations of NPs were examined by FT-IR, VSM, and SEM. Modified nanoparticles with average diameters of 20–80 nm were obtained. Enzyme immobilization efficiencies of 89 and 74 were obtained for AFSMNPs and chitosan-coated AFSMNPs, respectively. The optimum pH obtained was 6.5 and 8.0 for the enzyme immobilized on AFSMNPs and chitosan-coated AFSMNPs, respectively. Optimum temperature for the immobilized enzyme shifted toward higher temperatures. Considerable enhancements in thermal stabilities were observed for the immobilized enzyme at elevated temperatures up to 80 °C. A frequent use experiment demonstrated that the immobilized enzyme retained 74 and 85 % of its original activity even after 20 times of repeated use in AFSMNPs and chitosan-coated AFSMNPs, respectively. Storage stability demonstrated that free enzyme lost its activity completely within 30 days. But, immobilized enzyme on AFSMNPs and chitosan-coated AFSMNPs preserved 65.73 and 78.63 % of its initial activity, respectively, after 80 days of incubation. In conclusion, a substantial improvement in the performance of the immobilized enzyme with reference to the free enzyme was obtained. Furthermore, the relative activities of immobilized enzyme are superior than free enzyme over the broader pH and temperature ranges.

Immobilization of poly(MPC) brushes onto titanium surface by combining dopamine self-polymerization and ATRP: Preparation, characterization and evaluation of hemocompatibility in vitro

Energy Technology Data Exchange (ETDEWEB)

Ma, Wenyong; Yang, Ping; Li, Jingan; Li, Shiqi; Li, Peichuang; Zhao, Yuancong, E-mail: Zhaoyc7320@163.com; Huang, Nan

2015-09-15

Graphical abstract: The functional surface containing rich amino and hydroxyl groups was obtained by simple and easily dopamine self-polymerization. Poly (MPC) brushes were successfully immobilized on titanium surface by combining acylation reaction and ATRP. This chemical and biomimetic modified titanium surface effectively inhibits platelet adhesion and activation. - Highlights: • Polydopamine coating provides amino and hydroxyl groups for second reactivity. • Poly(MPC) brushes covalently immobilized on coating by surface initiated ATRP. • In vitro hemocompatibility of biomimetic modified Ti was better than unmodified. - Abstract: Poly(2-methacryloyloxyethyl phosphorylcholine(MPC)) has been studied in many biomedical fields because of good biocompatibility, such as hemocompatibility, inhibiting protein adhesion, antifouling, and so on. To achieve good hemocompatibility of titanium (Ti) surface, bio-inspired poly(MPC) brushes were grafted from Ti substrate covalently. In this work, the surface of Ti was first coated with polydopamine, and got a surface bearing −NH{sub 2} and −OH groups which could be bridged with poly(MPC) via atom transfer radical polymerization. Water contact angle decreased to 51.3° when titanium grafted with poly(MPC) brushes. The data of Infrared Spectroscopy and X-ray photoelectron spectroscopy (XPS) indicated that poly(MPC) was successfully grafted onto the surface of titanium. Platelet-rich plasma (PRP) adhesion test and lactate dehydrogenase (LDH) assay showed that the number of platelets adhered on the surface of modified-titanium was much less than that of unmodified titanium and platelets did not aggregate and distort. Thus, the simple and chemical method of immobilization of poly(MPC) brushes has potential application for improving hemocompatibility for cardiovascular stent and some other biomaterials.

Physico-chemical, spectroscopical and thermal characterization of bio diesel obtained by enzymatic route as a tool to select the most efficient immobilized lipase

International Nuclear Information System (INIS)

Silva, G.A.M.; Ros, P.C.M. da; Souza, L.T.A.; Costa, A.P.O.; Castro, H.F. de

2012-01-01

Two microbial lipases from Burkholderia cepacia and Pseudomonas fluorescens were evaluated as catalysts for the enzymatic transesterification of beef tallow with ethanol and the most efficient lipase source was selected by taking into account the properties of the product to be used as fuel. Both lipases were immobilized on an epoxy silica-polyvinyl alcohol composite by covalent immobilization and used to perform the reactions under the following operational conditions: beef tallow-to-ethanol molar ratio of 1:9, 45 degree C and 400 units of enzymatic activity per gram of fat. Products, characterized using Fourier Transform Infrared spectroscopy (FTIR), viscosimetry, thermogravimetry and 1 H NMR spectroscopy, suggested that the bio diesel sample obtained in the reaction catalyzed by Burkholderia cepacia lipase has the best set of properties for fuel usage. (author)

Physico-chemical, spectroscopical and thermal characterization of bio diesel obtained by enzymatic route as a tool to select the most efficient immobilized lipase

Energy Technology Data Exchange (ETDEWEB)

Silva, G.A.M.; Ros, P.C.M. da; Souza, L.T.A.; Costa, A.P.O.; Castro, H.F. de, E-mail: heizir@dequi.eel.usp.br [Engineering School of Lorena. University of Sao Paulo (EEL/USP), Lorena, SP (Brazil)

2012-01-15

Two microbial lipases from Burkholderia cepacia and Pseudomonas fluorescens were evaluated as catalysts for the enzymatic transesterification of beef tallow with ethanol and the most efficient lipase source was selected by taking into account the properties of the product to be used as fuel. Both lipases were immobilized on an epoxy silica-polyvinyl alcohol composite by covalent immobilization and used to perform the reactions under the following operational conditions: beef tallow-to-ethanol molar ratio of 1:9, 45 degree C and 400 units of enzymatic activity per gram of fat. Products, characterized using Fourier Transform Infrared spectroscopy (FTIR), viscosimetry, thermogravimetry and {sup 1}H NMR spectroscopy, suggested that the bio diesel sample obtained in the reaction catalyzed by Burkholderia cepacia lipase has the best set of properties for fuel usage. (author)

Theory and Applications of Covalent Docking in Drug Discovery: Merits and Pitfalls

Directory of Open Access Journals (Sweden)

Hezekiel Mathambo Kumalo

2015-01-01

Full Text Available he present art of drug discovery and design of new drugs is based on suicidal irreversible inhibitors. Covalent inhibition is the strategy that is used to achieve irreversible inhibition. Irreversible inhibitors interact with their targets in a time-dependent fashion, and the reaction proceeds to completion rather than to equilibrium. Covalent inhibitors possessed some significant advantages over non-covalent inhibitors such as covalent warheads can target rare, non-conserved residue of a particular target protein and thus led to development of highly selective inhibitors, covalent inhibitors can be effective in targeting proteins with shallow binding cleavage which will led to development of novel inhibitors with increased potency than non-covalent inhibitors. Several computational approaches have been developed to simulate covalent interactions; however, this is still a challenging area to explore. Covalent molecular docking has been recently implemented in the computer-aided drug design workflows to describe covalent interactions between inhibitors and biological targets. In this review we highlight: (i covalent interactions in biomolecular systems; (ii the mathematical framework of covalent molecular docking; (iii implementation of covalent docking protocol in drug design workflows; (iv applications covalent docking: case studies and (v shortcomings and future perspectives of covalent docking. To the best of our knowledge; this review is the first account that highlights different aspects of covalent docking with its merits and pitfalls. We believe that the method and applications highlighted in this study will help future efforts towards the design of irreversible inhibitors.

Immobilization of microorganisms. Part 1. Preparation of immobilized Lactobacillus bulgaricus

Energy Technology Data Exchange (ETDEWEB)

Lee, K H

1981-01-01

The immobilization of Lactobacillus bulgaricus on polyacrylamide and on alginate beads was investigated. The most active immobilized cells were obtained by entrapment in Ca alginate beads. These immobilized microbial cells, when introduced into 4.5% lactose solution and whey solution showed maximum relative activity of 28% for lactose and 18% for whey compared to free cells.

Production and action pattern of inulinase from Aspergillus Niger-245: hydrolysis of inulin from several sources

Directory of Open Access Journals (Sweden)

Cruz Vinícius D?Arcadia

1998-01-01

Full Text Available A strain of Aspergillus niger isolated from soil samples showed great capacity to produce extracellular inulinase. Although the enzyme has been synthesized in presence of monosaccharides, sucrose and sugar cane molasse, the productivity was significantly higher (p<0.05 when the microorganism was inoculated in media formulated with dahlia extract and pure inulin, as carbon sources. With regard to the nitrogen source, the best results were obtained with casein and other sources of proteic nitrogen, comparatively to the mineral nitrogen. However, statistic significance (p<0.01 only was found between the productivity obtained in the medium prepared with casein and ammonium sulphate. The optimum pH of the purified enzyme for inulin hydrolysis was found between 4.0 and 4.5 and the optimun temperature at 60oC. When treated by 30 minutes in this temperature no loss of activity was observed. The enzyme showed capacity to hydrolyse sucrose, raffinose and inulin from which it liberated only fructose units showing, therefore, an exo-action mechanism. Acting on inulins from several sources, the enzyme showed larger hydrolysis speed on the polissaccharide from chicory (Cichorium intibus, comparatively, to the inulins from dahlia (Dahlia pinnata and Jerusalem artichoke (Helianthus tuberosus roots.

Mechanical desorption of immobilized proteins using carbon dioxide aerosols for reusable biosensors

Energy Technology Data Exchange (ETDEWEB)

Singh, Renu; Hong, Seongkyeol [School of Mechanical and Nuclear Engineering, Ulsan National Institute of Science and Technology (UNIST), Ulsan 689-798 (Korea, Republic of); Jang, Jaesung, E-mail: jjang@unist.ac.kr [School of Mechanical and Nuclear Engineering, Ulsan National Institute of Science and Technology (UNIST), Ulsan 689-798 (Korea, Republic of); Department of Biomedical Engineering, Ulsan National Institute of Science and Technology (UNIST), Ulsan 689-798 (Korea, Republic of); School of Materials Science and Engineering, Ulsan National Institute of Science and Technology (UNIST), Ulsan 689-798 (Korea, Republic of)

2015-01-01

Highlights: • Immobilized proteins were removed using carbon dioxide aerosols. • We observed high removal efficiencies due to the aerosol treatment. • We confirmed the removal with FTIR and X-ray photoelectron spectroscopy. • This CO{sub 2} aerosol treatment did not undermine re-functionalization. • This technique is a fast and damage-free method to reuse a sensor surface. - Abstract: Reusability of a biosensor has recently received considerable attention, and it is closely related with the effective desorption of probe molecules. We present a novel mechanical desorption technique to reuse biosensors by using periodic jets of carbon dioxide (CO{sub 2}) aerosols (a mixture of solid and gaseous CO{sub 2}), and demonstrate its feasibility by removing physically adsorbed and covalently bonded fluorescent proteins i.e., Escherichia coli fluorescein isothiocyanate antibody and bovine serum albumin (E. coli FITC–Ab and FITC–BSA) from silicon chips. The proteins on the chip surfaces were measured by fluorescent images before and after applying the aerosols. The removal efficiency of the aerosol treatment was measured for various concentrations (1–20 μg mL{sup −1}) of E. coli FITC–Ab and FITC–BSA with two different removal cycles (5 and 11 cycles; each cycle: 8 s). We observed high removal efficiencies (>93.5% for physically adsorbed Ab and >84.6% for covalently bonded Ab) at 11 cycle aerosol treatment. This CO{sub 2} aerosol treatment did not undermine re-functionalization, which was confirmed by the fluorescent images of FITC–Abs for fresh and reused chips. Desorption of the immobilized layers was validated by Fourier transform infrared and X-ray photoelectron spectroscopic analyses. We also conducted an experiment on the regeneration of E. coli sensing chips using this aerosol treatment, and the chips were re-used 5 times successfully. This mechanical desorption technique is a highly effective and novel strategy for reusable biosensors.

Determinação das propriedades catalíticas em meio aquoso e orgânico da lipase de Candida rugosa imobilizada em celulignina quimicamente modificada por carbonildiimidazol Assessment of catalytic properties in aqueous and organic media of lipase from Candida rugosa immobilized on wood cellulignin activated with carbonyldiimidazole

Directory of Open Access Journals (Sweden)

Fabrício M. Gomes

2006-07-01

Full Text Available Microbial lipase from Candida rugosa was immobilized by covalent binding on wood cellulignin (Eucaliptus grandis chemically modified with carbonyldiimidazole. The immobilized system was fully evaluated in aqueous (olive oil hydrolysis and organic (ester synthesis media. A comparative study between free and immobilized lipase was carried out in terms of pH, temperature and thermal stability. A higher pH value (8.0 was found optimal for the immobilized lipase. The optimal reaction temperature shifted from 37 °C for the free lipase to 45 °C for the immobilized lipase. The pattern of heat stability indicated that the immobilization process tends to stabilize the enzyme. Kinetics tests at 37 °C following the hydrolysis of olive oil obeyed the Michaelis-Menten rate equation. Values for Km = 924.9 mM and Vmax = 198.3 U/mg were lower than for free lipase, suggesting that the affinity towards the substrate changed and the activity of the immobilized lipase decreased during the course of immobilization. The immobilized derivative was also tested in the ester synthesis from several alcohols and carboxylic acids.

Study of the luminescence of tris(2-thenoyltrifluoroacetonato)lanthanide(III) complexes covalently linked to 1,10-phenanthroline-functionalized hybrid sol-gel glasses

International Nuclear Information System (INIS)

Lenaerts, Philip; Ryckebosch, Eline; Driesen, Kris; Deun, Rik van; Nockemann, Peter; Goerller-Walrand, Christiane; Binnemans, Koen

2005-01-01

The solubility and uniform distribution of lanthanide complexes in sol-gel glasses can be improved by covalently linking the complexes to the sol-gel matrix. In this study, several lanthanide β-diketonate complexes (Ln=Nd, Sm, Eu, Tb, Er, Yb) were immobilized on a 1,10-phenanthroline functionalized sol-gel glass. For the europium(III) complex, a sol-gel material of diethoxydimethylsilane (DEDMS) with polymer-like properties was derived. For the other lanthanide complexes, the sol-gel glass was prepared by using a matrix of tetramethoxysilane (TMOS) and DEDMS. Both systems were prepared under neutral reaction conditions. High-resolution emission and excitation spectra were recorded. The luminescence lifetimes were measured

Recent Advances in Immobilization Strategies for Glycosidases

Science.gov (United States)

Karav, Sercan; Cohen, Joshua L.; Barile, Daniela; de Moura Bell, Juliana Maria Leite Nobrega

2017-01-01

Glycans play important biological roles in cell-to-cell interactions, protection against pathogens, as well as in proper protein folding and stability, and are thus interesting targets for scientists. Although their mechanisms of action have been widely investigated and hypothesized, their biological functions are not well understood due to the lack of deglycosylation methods for large-scale isolation of these compounds. Isolation of glycans in their native state is crucial for the investigation of their biological functions. However, current enzymatic and chemical deglycosylation techniques require harsh pretreatment and reaction conditions (high temperature and use of detergents) that hinder the isolation of native glycan structures. Indeed, the recent isolation of new endoglycosidases that are able to cleave a wider variety of linkages and efficiently hydrolyze native proteins has opened up the opportunity to elucidate the biological roles of a higher variety of glycans in their native state. As an example, our research group recently isolated a novel Endo-β-N-acetylglucosaminidase from Bifidobacterium longum subsp. infantis ATCC 15697 (EndoBI-1) that cleaves N-N′-diacetyl chitobiose moieties found in the N-linked glycan (N-glycan) core of high mannose, hybrid, and complex N-glycans. This enzyme is also active on native proteins, which enables native glycan isolation, a key advantage when evaluating their biological activities. Efficient, stable, and economically viable enzymatic release of N-glycans requires the selection of appropriate immobilization strategies. In this review, we discuss the state-of-the-art of various immobilization techniques (physical adsorption, covalent binding, aggregation, and entrapment) for glycosidases, as well as their potential substrates and matrices. PMID:27718339

Enhanced performance of a glucose/O(2) biofuel cell assembled with laccase-covalently immobilized three-dimensional macroporous gold film-based biocathode and bacterial surface displayed glucose dehydrogenase-based bioanode.

Science.gov (United States)

Hou, Chuantao; Yang, Dapeng; Liang, Bo; Liu, Aihua

2014-06-17

The power output and stability of enzyme-based biofuel cells (BFCs) is greatly dependent on the properties of both the biocathode and bioanode, which may be adapted for portable power production. In this paper, a novel highly uniform three-dimensional (3D) macroporous gold (MP-Au) film was prepared by heating the gold "supraspheres", which were synthesized by a bottom-up protein templating approach, and followed by modification of laccase on the MP-Au film by covalent immobilization. The as-prepared laccase/MP-Au biocathode exihibited an onset potential of 0.62 V versus saturated calomel electrode (SCE, or 0.86 V vs NHE, normal hydrogen electrode) toward O2 reduction and a high catalytic current of 0.61 mAcm(-2). On the other hand, mutated glucose dehydrogenase (GDH) surface displayed bacteria (GDH-bacteria) were used to improve the stability of the glucose oxidation at the bioanode. The as-assembled membraneless glucose/O2 fuel cell showed a high power output of 55.8 ± 2.0 μW cm(-2) and open circuit potential of 0.80 V, contributing to the improved electrocatalysis toward O2 reduction at the laccase/MP-Au biocathode. Moreover, the BFC retained 84% of its maximal power density even after continuous operation for 55 h because of the high stability of the bacterial surface displayed GDH mutant toward glucose oxidation. Our findings may be promising for the development of more efficient glucose BFC for portable battery or self-powered device applications.

Atomic Covalent Functionalization of Graphene

Science.gov (United States)

Johns, James E.; Hersam, Mark C.

2012-01-01

Conspectus Although graphene’s physical structure is a single atom thick, two-dimensional, hexagonal crystal of sp2 bonded carbon, this simple description belies the myriad interesting and complex physical properties attributed to this fascinating material. Because of its unusual electronic structure and superlative properties, graphene serves as a leading candidate for many next generation technologies including high frequency electronics, broadband photodetectors, biological and gas sensors, and transparent conductive coatings. Despite this promise, researchers could apply graphene more routinely in real-world technologies if they could chemically adjust graphene’s electronic properties. For example, the covalent modification of graphene to create a band gap comparable to silicon (~1 eV) would enable its use in digital electronics, and larger band gaps would provide new opportunities for graphene-based photonics. Towards this end, researchers have focused considerable effort on the chemical functionalization of graphene. Due to its high thermodynamic stability and chemical inertness, new methods and techniques are required to create covalent bonds without promoting undesirable side reactions or irreversible damage to the underlying carbon lattice. In this Account, we review and discuss recent theoretical and experimental work studying covalent modifications to graphene using gas phase atomic radicals. Atomic radicals have sufficient energy to overcome the kinetic and thermodynamic barriers associated with covalent reactions on the basal plane of graphene but lack the energy required to break the C-C sigma bonds that would destroy the carbon lattice. Furthermore, because they are atomic species, radicals substantially reduce the likelihood of unwanted side reactions that confound other covalent chemistries. Overall, these methods based on atomic radicals show promise for the homogeneous functionalization of graphene and the production of new classes of two

Covalent Surface Modifications of Carbon Nanotubes.

Energy Technology Data Exchange (ETDEWEB)

Pavia Sanders, Adriana [Sandia National Lab. (SNL-CA), Livermore, CA (United States); O' Bryan, Greg [Sandia National Lab. (SNL-CA), Livermore, CA (United States)

2017-07-01

A report meant to document the chemistries investigated by the author for covalent surface modification of CNTs. Oxidation, cycloaddition, and radical reactions were explored to determine their success at covalently altering the CNT surface. Characterization through infrared spectroscopy, Raman spectroscopy, and thermo gravimetric analysis was performed in order to determine the success of the chemistries employed. This report is not exhaustive and was performed for CNT surface modification exploration as it pertains to the "Next Gen" project.

The nature of the Iron Moiety bisorped by immobilized Saccharomyces Cervisiae at low pH: A Mossbauer spectroscopic investigation

International Nuclear Information System (INIS)

Khalil, Mustaim I.; Al-Wassil, Abdulaziz I.

1999-01-01

The nature of the adsorped Fe-moiety on immobilized Saccharomyces Cervisiae at low pH has been investigated by Mossbauer spectroscopy. The Mossbauer spectrum at 77K exhibited two sites: the major one (69%) was a quadrupole-split double, Delta Q=0.77 mms with an isomer shift 0.46 mms, assigned to the high spin octahedrally coordinated iron (III); and a single line minor site (31%) with an isomer shift, d=0.36 mms, assigned to the high-spin tetrahedral iron (III) Cl-moiety. An electrostatic and a covalent mode of Fe binding were then inferred. (author)

Concanavalin A immobilized magnetic poly(glycidyl methacrylate) beads for prostate specific antigen binding.

Science.gov (United States)

Idil, Neslihan; Perçin, Işık; Karakoç, Veyis; Yavuz, Handan; Aksöz, Nilüfer; Denizli, Adil

2015-10-01

The aim of this study was to prepare Concanavalin A (Con A) immobilized magnetic poly(glycidyl methacrylate) (mPGMA) beads for prostate specific antigen (PSA) binding and to study binding capacities of the beads using lectin-glycoprotein interactions. Firstly, iron oxide nanoparticles were synthesized by co-precipitation method and then, beads were synthesized by dispersion polymerization in the presence of iron oxide nanoparticles. Con A molecules were both covalently immobilized onto the beads directly and through the spacer arm (1,6-diaminohexane-HDMA). The total PSA and free PSA binding onto the mPGMA-HDMA-Con A beads were higher than that of the mPGMA-Con A beads. Maximum PSA binding capacity was observed as 91.2 ng/g. Approximately 45% of the bound PSA was eluted by using 0.1 M mannose as elution agent. The mPGMA-HDMA-Con A beads could be reused without a remarkable decrease in the binding capacities after 5 binding-desorption cycles. Serum fractions were analyzed using SDS-PAGE. The mPGMA-HDMA-Con A beads could be useful for the detection of PSA and suggested as a model system for other glycoprotein biomarkers. Copyright © 2015 Elsevier B.V. All rights reserved.

Immobilization of Aspergillus niger. beta. -D-glucosidase on aminated chitin and alumina/alginate

Energy Technology Data Exchange (ETDEWEB)

Bon, E.; Freire, D.; Mendes, M.F.; Soares. V.F.

1986-01-01

The immobilization of ..beta..-glucosidase was studied by (a) covalent coupling to aminated chitin (IME-C) and (b) adsorption onto alumina followed by gel entrapment of the suspension with calcium alginate (IME-A). The levels of catalytic activity determined against salicin at 50 C were 23.0 U/g and 0.2 U/g for the IME-C and IMA-A respectively. The first system was shown to be quite stable with a loss of only 2% of the initial activity over 14 days. The IME-A system had a half life of 14 days. The activity of IME-C was studied using cellobiose and enzymatic hydrolysates of sugar cane bagasse at several cellobiose concentrations. The activities obtained with cellobiose were 104.0 U/g and 72.0 U/g respectively. 13 references.

Immobilization of cholesterol esterase and cholesterol oxidase onto sol-gel films for application to cholesterol biosensor

International Nuclear Information System (INIS)

Singh, Suman; Singhal, Rahul; Malhotra, B.D.

2007-01-01

Cholesterol oxidase (ChOx) and cholesterol esterase (ChEt) have been covalently immobilized onto tetraethylorthosilicate (TEOS) sol-gel films. The tetraethylorthosilicate sol-gel/ChEt/ChOx enzyme films thus prepared have been characterized using scanning electron microscopic (SEM), UV-vis spectroscopic, Fourier-transform-infrared (FTIR) spectroscopic and amperometric techniques, respectively. The results of photometric measurements carried out on tetraethylorthosilicate sol-gel/ChEt/ChOx reveal thermal stability up to 55 deg. C, response time as 180 s, linearity up to 780 mg dL -1 (12 mM), shelf life of 1 month, detection limit of 12 mg dL -1 and sensitivity as 5.4 x 10 -5 Abs. mg -1 dL -1

Limb immobilization and corticobasal syndrome.

Science.gov (United States)

Graff-Radford, Jonathan; Boeve, Bradley F; Drubach, Daniel A; Knopman, David S; Ahlskog, J Eric; Golden, Erin C; Drubach, Dina I; Petersen, Ronald C; Josephs, Keith A

2012-12-01

Recently, we evaluated two patients with corticobasal syndrome (CBS) who reported symptom onset after limb immobilization. Our objective was to investigate the association between trauma, immobilization and CBS. The charts of forty-four consecutive CBS patients seen in the Mayo Clinic Alzheimer Disease Research Center were reviewed with attention to trauma and limb immobilization. 10 CBS patients (23%) had immobilization or trauma on the most affected limb preceding the onset or acceleration of symptoms. The median age at onset was 61. Six patients manifested their first symptoms after immobilization from surgery or fracture with one after leg trauma. Four patients had pre-existing symptoms of limb dysfunction but significantly worsened after immobilization or surgery. 23 percent of patients had immobilization or trauma of the affected limb. This might have implications for management of CBS, for avoiding injury, limiting immobilization and increasing movement in the affected limb. Copyright © 2012 Elsevier Ltd. All rights reserved.

Highly sensitive covalently functionalized light-addressable potentiometric sensor for determination of biomarker

Energy Technology Data Exchange (ETDEWEB)

Liang, Jintao [School of Life and Environmental Sciences, Guilin University of Electronic Technology, Guilin, Guangxi 541004 (China); Guangxi Experiment Center of Information Sciences, Guilin University of Electronic Technology, Guilin, Guangxi 541004 (China); Guan, Mingyuan; Huang, Guoyin; Qiu, Hengming; Chen, Zhengcheng [School of Life and Environmental Sciences, Guilin University of Electronic Technology, Guilin, Guangxi 541004 (China); Li, Guiyin, E-mail: liguiyin01@163.com [School of Life and Environmental Sciences, Guilin University of Electronic Technology, Guilin, Guangxi 541004 (China); Guangxi Experiment Center of Information Sciences, Guilin University of Electronic Technology, Guilin, Guangxi 541004 (China); Huang, Yong, E-mail: huangyong503@126.com [School of Life and Environmental Sciences, Guilin University of Electronic Technology, Guilin, Guangxi 541004 (China); Guangxi Experiment Center of Information Sciences, Guilin University of Electronic Technology, Guilin, Guangxi 541004 (China)

2016-06-01

A biomarker is related to the biological status of a living organism and shows great promise for the early prediction of a related disease. Herein we presented a novel structured light-addressable potentiometric sensor (LAPS) for the determination of a model biomarker, human immunoglobulin G (hIgG). In this system, the goat anti-human immunoglobulin G antibody was used as recognition element and covalently immobilized on the surface of light-addressable potentiometric sensor chip to capture human immunoglobulin G. Due to the light addressable capability of light-addressable potentiometric sensor, human immunoglobulin G dissolved in the supporting electrolyte solution can be detected by monitoring the potential shifts of the sensor. In order to produce a stable photocurrent, the laser diode controlled by field-programmable gate array was used as the light emitter to drive the light-addressable potentiometric sensor. A linear correlation between the potential shift response and the concentration of human immunoglobulin G was achieved and the corresponding regression equation was ΔV (V) = 0.00714C{sub hIgG} (μg/mL)–0.0147 with a correlation coefficient of 0.9968 over a range 0–150 μg/mL. Moreover, the light-addressable potentiometric sensor system also showed acceptable stability and reproducibility. All the results demonstrated that the system was more applicable to detection of disease biomarkers with simple operation, multiple-sample format and might hold great promise in various environmental, food, and clinical applications. - Highlights: • A novel structured light-addressable potentiometric sensor (LAPS) based on covalently functionalized membrane was designed. • The composition of the surface of LAPS chip was investigated by X-ray photoelectron spectroscopy (XPS). • hIgG dissolved in the supporting electrolyte solution can be detected by monitoring the potential shifts of LAPS.

Fabrication of graphene oxide decorated with Fe{sub 3}O{sub 4}@SiO{sub 2} for immobilization of cellulase

Energy Technology Data Exchange (ETDEWEB)

Li, Yue; Wang, Xiang-Yu; Jiang, Xiao-Ping; Ye, Jing-Jing; Zhang, Ye-Wang, E-mail: zhangyewang@ujs.edu.cn [Jiangsu University, School of Pharmacy (China); Zhang, Xiao-Yun, E-mail: zhangxiaoyungu@126.com [Jiangsu University, School of Food and Biological Engineering (China)

2015-01-15

Fe{sub 3}O{sub 4}@SiO{sub 2}–graphene oxide (GO) composites were successfully fabricated by chemical binding of functional Fe{sub 3}O{sub 4}@SiO{sub 2} and GO and applied to immobilization of cellulase via covalent attachment. The prepared composites were further characterized by transmission electron microscopy and Fourier transform infrared spectroscopy. Fe{sub 3}O{sub 4} nanoparticles (NPs) were monodisperse spheres with a mean diameter of 17 ± 0.2 nm. The thickness of SiO{sub 2} layer was calculated as being 6.5 ± 0.2 nm. The size of Fe{sub 3}O{sub 4}@SiO{sub 2} NPs was 24 ± 0.3 nm, similar to that of Fe{sub 3}O{sub 4}@SiO{sub 2}–NH{sub 2}. Fe{sub 3}O{sub 4}@SiO{sub 2}–GO composites were synthesized by linking of Fe{sub 3}O{sub 4}@SiO{sub 2}–NH{sub 2} NPs to GO with the catalysis of EDC and NHS. The prepared composites were used for immobilization of cellulase. A high immobilization yield and efficiency of above 90 % were obtained after the optimization. The half-life of immobilized cellulase (722 min) was 3.34-fold higher than that of free enzyme (216 min) at 50 °C. Compared with the free cellulase, the optimal temperature of the immobilized enzyme was not changed; but the optimal pH was shifted from 5.0 to 4.0, and the thermal stability was enhanced. The immobilized cellulase could be easily separated and reused under magnetic field. These results strongly indicate that the cellulase immobilized onto the Fe{sub 3}O{sub 4}@SiO{sub 2}–GO composite has potential applications in the production of bioethanol.

Status of plutonium ceramic immobilization processes and immobilization forms

International Nuclear Information System (INIS)

Ebbinghaus, B.B.; Van Konynenburg, R.A.; Vance, E.R.; Jostsons, A.

1996-01-01

Immobilization in a ceramic followed by permanent emplacement in a repository or borehole is one of the alternatives currently being considered by the Fissile Materials Disposition Program for the ultimate disposal of excess weapons-grade plutonium. To make Pu recovery more difficult, radioactive cesium may also be incorporated into the immobilization form. Valuable data are already available for ceramics form R ampersand D efforts to immobilize high-level and mixed wastes. Ceramics have a high capacity for actinides, cesium, and some neutron absorbers. A unique characteristic of ceramics is the existence of mineral analogues found in nature that have demonstrated actinide immobilization over geologic time periods. The ceramic form currently being considered for plutonium disposition is a synthetic rock (SYNROC) material composed primarily of zirconolite (CaZrTi 2 O 7 ), the desired actinide host phase, with lesser amounts of hollandite (BaAl 2 Ti 6 O 16 ) and rutile (TiO 2 ). Alternative actinide host phases are also being considered. These include pyrochlore (Gd 2 Ti 2 O 7 ), zircon (ZrSiO 4 ), and monazite (CePO 4 ), to name a few of the most promising. R ampersand D activities to address important technical issues are discussed. Primarily these include moderate scale hot press fabrications with plutonium, direct loading of PuO 2 powder, cold press and sinter fabrication methods, and immobilization form formulation issues

Covalent versus ionic bonding in alkalimetal fluoride oligomers

NARCIS (Netherlands)

Bickelhaupt, F.M.; Sola, M.; Fonseca Guerra, C.

2007-01-01

The most polar bond in chemistry is that between a fluorine and an alkalimetal atom. Inspired by our recent finding that other polar bonds (C - M and H - M) have important covalent contributions (i.e., stabilization due to bond overlap), we herein address the question if covalency is also essential

Two supramolecular complexes based on polyoxometalates and Co-EDTA units via covalent connection or non-covalent interaction

Energy Technology Data Exchange (ETDEWEB)

Teng, Chunlin; Xiao, Hanxi [Key Laboratory of Theoretical Organic Chemistry and Functional Molecule for Ministry of Education, Hunan University of Science and Technology, Xiangtan 411201 (China); Cai, Qing [Chemistry Department, City University of New York, New York, NY 10016 (United States); Tang, Jianting; Cai, Tiejun [Key Laboratory of Theoretical Organic Chemistry and Functional Molecule for Ministry of Education, Hunan University of Science and Technology, Xiangtan 411201 (China); Deng, Qian, E-mail: dengqian10502@163.com [Key Laboratory of Theoretical Organic Chemistry and Functional Molecule for Ministry of Education, Hunan University of Science and Technology, Xiangtan 411201 (China)

2016-11-15

Two new 3D network organic-inorganic hybrid supramolecular complexes ([Na{sub 6}(CoEDTA){sub 2}(H{sub 2}O){sub 13}]·(H{sub 2}SiW{sub 12}O{sub 40})·xH{sub 2}O)n (1) and [CoH{sub 4}EDTA(H{sub 2}O)]{sub 2}(SiW{sub 12}O{sub 40})·15H{sub 2}O (2) (H{sub 4}EDTA=Ethylenediamine tetraacetic acid) have been successfully synthesized by solution method, and characterized by infrared spectrum (IR), thermogravimetric-differential thermal analysis (TG-DTA), cyclic voltammetry (CV) and single{sup −}crystal X-ray diffraction (XRD). Both of the complexes are the supramolecules, but with different liking mode, they are two representative models of supramolecule. complex (1) is a 3D infinite network supramolecular coordination polymer with a rare multi-metal sturcture of sodium-cobalt-containing, which is mainly linked through coordinate-covalent bonds. While complex (2) is normal supramolecule, which linked by non-covalent interactions, such as H-bonding interaction, electrostatic interaction and van der waals force. Both of complex (1) and (2) exhibit good catalytic activities for catalytic oxidation of methanol, when the initial concentration of methanol is 3.0 g m{sup −3}, flow rate is 10 mL min{sup −1}, and the quality of catalyst is 0.2 g, for complex (1) and complex (2) the maximum elimination rates of methanol are 85% (150 °C) and 92% (120 °C), respectively. - Graphical abstract: Two new organic-inorganic hybrid supramolecular complexes based on Co-EDTA, and Keggin polyanions have been successfully synthesized with different pH value by solution method. They are attributed to two representative models of supramolecule. Complex(1) is an infinite coordination polymer with a rare multi-metal sturcture of sodium-cobalt-containing, which is mainly linked through covalent bonds. Complex (2) is a normal supramolecule, which linked by non-covalent interactions of H-bonding interaction, electrostatic interaction and van der waals force. - Highlights: • Two supramolecules

Boronic Acid-Based Approach for Separation and Immobilization of Glycoproteins and Its Application in Sensing

Directory of Open Access Journals (Sweden)

Lin Liu

2013-10-01

Full Text Available Glycoproteins influence a broad spectrum of biological processes including cell-cell interaction, host-pathogen interaction, or protection of proteins against proteolytic degradation. The analysis of their glyco-structures and concentration levels are increasingly important in diagnosis and proteomics. Boronic acids can covalently react with cis-diols in the oligosaccharide chains of glycoproteins to form five- or six-membered cyclic esters. Based on this interaction, boronic acid-based ligands and materials have attracted much attention in both chemistry and biology as the recognition motif for enrichment and chemo/biosensing of glycoproteins in recent years. In this work, we reviewed the progress in the separation, immobilization and detection of glycoproteins with boronic acid-functionalized materials and addressed its application in sensing.

Mesoporous silicas with covalently immobilized β-cyclodextrin moieties: synthesis, structure, and sorption properties

Science.gov (United States)

Roik, Nadiia V.; Belyakova, Lyudmila A.; Trofymchuk, Iryna M.; Dziazko, Marina O.; Oranska, Olena I.

2017-09-01

Mesoporous silicas with chemically attached macrocyclic moieties were successfully prepared by sol-gel condensation of tetraethyl orthosilicate and β-cyclodextrin-silane in the presence of a structure-directing agent. Introduction of β-cyclodextrin groups into the silica framework was confirmed by the results of IR spectral, thermogravimetric, and quantitative chemical analysis of surface compounds. The porous structure of the obtained materials was characterized by nitrogen adsorption-desorption measurements, powder X-ray diffraction, transmission electron microscopy, and dynamic light scattering. It was found that the composition of the reaction mixture used in β-cyclodextrin-silane synthesis significantly affects the structural parameters of the resulting silicas. The increase in (3-aminopropyl)triethoxysilane as well as the coupling agent content in relation to β-cyclodextrin leads ultimately to the lowering or complete loss of hexagonal arrangement of pore channels in the synthesized materials. Formation of hexagonally ordered mesoporous structure was observed at molar composition of the mixture 0.049 TEOS:0.001 β-CD-silane:0.007 CTMAB:0.27 NH4OH:7.2 H2O and equimolar ratio of components in β-CD-silane synthesis. The sorption of alizarin yellow on starting silica and synthesized materials with chemically attached β-cyclodextrin moieties was studied in phosphate buffer solutions with pH 7.0. Experimental results of the dye equilibrium sorption were analyzed using Langmuir, Freundlich, and Redlich-Peterson isotherm models. It was proved that the Redlich-Peterson isotherm model is the most appropriate for fitting the equilibrium sorption of alizarin yellow on parent silica with hexagonally arranged mesoporous structure as well as on modified one with chemically immobilized β-cyclodextrin groups. [Figure not available: see fulltext.

Covalent modification and exfoliation of graphene oxide using ferrocene

Science.gov (United States)

Avinash, M. B.; Subrahmanyam, K. S.; Sundarayya, Y.; Govindaraju, T.

2010-09-01

Large scale preparation of single-layer graphene and graphene oxide is of great importance due to their potential applications. We report a simple room temperature method for the exfoliation of graphene oxide using covalent modification of graphene oxide with ferrocene to obtain single-layer graphene oxide sheets. The samples were characterized by FESEM, HRTEM, AFM, EDAX, FT-IR, Raman and Mössbauer spectroscopic studies. HRTEM micrograph of the covalently modified graphene oxide showed increased interlayer spacing of ~2.4 nm due to ferrocene intercalation. The presence of single-layer graphene oxide sheets were confirmed by AFM studies. The covalently modified ferrocene-graphene oxide composite showed interesting magnetic behavior.Large scale preparation of single-layer graphene and graphene oxide is of great importance due to their potential applications. We report a simple room temperature method for the exfoliation of graphene oxide using covalent modification of graphene oxide with ferrocene to obtain single-layer graphene oxide sheets. The samples were characterized by FESEM, HRTEM, AFM, EDAX, FT-IR, Raman and Mössbauer spectroscopic studies. HRTEM micrograph of the covalently modified graphene oxide showed increased interlayer spacing of ~2.4 nm due to ferrocene intercalation. The presence of single-layer graphene oxide sheets were confirmed by AFM studies. The covalently modified ferrocene-graphene oxide composite showed interesting magnetic behavior. Electronic supplementary information (ESI) available: Magnetic data; AFM images; TEM micrographs; and Mössbauer spectroscopic data. See DOI: 10.1039/c0nr00024h

Status of plutonium ceramic immobilization processes and immobilization forms

Energy Technology Data Exchange (ETDEWEB)

Ebbinghaus, B.B.; Van Konynenburg, R.A. [Lawrence Livermore National Lab., CA (United States); Vance, E.R.; Jostsons, A. [Australian Nuclear Science and Technology Organization, Menai (Australia)] [and others

1996-05-01

Immobilization in a ceramic followed by permanent emplacement in a repository or borehole is one of the alternatives currently being considered by the Fissile Materials Disposition Program for the ultimate disposal of excess weapons-grade plutonium. To make Pu recovery more difficult, radioactive cesium may also be incorporated into the immobilization form. Valuable data are already available for ceramics form R&D efforts to immobilize high-level and mixed wastes. Ceramics have a high capacity for actinides, cesium, and some neutron absorbers. A unique characteristic of ceramics is the existence of mineral analogues found in nature that have demonstrated actinide immobilization over geologic time periods. The ceramic form currently being considered for plutonium disposition is a synthetic rock (SYNROC) material composed primarily of zirconolite (CaZrTi{sub 2}O{sub 7}), the desired actinide host phase, with lesser amounts of hollandite (BaAl{sub 2}Ti{sub 6}O{sub 16}) and rutile (TiO{sub 2}). Alternative actinide host phases are also being considered. These include pyrochlore (Gd{sub 2}Ti{sub 2}O{sub 7}), zircon (ZrSiO{sub 4}), and monazite (CePO{sub 4}), to name a few of the most promising. R&D activities to address important technical issues are discussed. Primarily these include moderate scale hot press fabrications with plutonium, direct loading of PuO{sub 2} powder, cold press and sinter fabrication methods, and immobilization form formulation issues.

A covalent attraction between two molecular cation TTF·~+

Institute of Scientific and Technical Information of China (English)

WANG FangFang; WANG Yi; WANG BingQiang; WANG YinFeng; MA Fang; Li ZhiRu

2009-01-01

The optimized structure of the tetrathiafulvalence radical-cation dimer (TTF·~+-TTF·~+) with all-real frequencies is obtained at MP2/6-311G level,which exhibits the attraction between two molecular cation TTF·~+.The new attraction interaction is a 20-center-2-electron intermolecular covalent π/π bonding with a telescope shape.The covalent π/π bonding has the bonding energy of about-21 kcal·mol~(-1) and is concealed by the Coulombic repulsion between two TTF·~+ cations.This intermolecular covalent attraction also influences the structure of the TTF·~+ subunit,I.e.,its molecular plane is bent by an angle θ=5.6°.This work provides new knowledge on intermolecular interaction.

A covalent attraction between two molecular cation TTF·~+

Institute of Scientific and Technical Information of China (English)

无

2009-01-01

The optimized structure of the tetrathiafulvalence radical-cation dimer(TTF·+-TTF·+) with all-real frequencies is obtained at MP2/6-311G level,which exhibits the attraction between two molecular cation TTF·+.The new attraction interaction is a 20-center-2-electron intermolecular covalent π /π bonding with a telescope shape.The covalent π /π bonding has the bonding energy of about -21 kcal·mol-1 and is concealed by the Coulombic repulsion between two TTF·+ cations.This intermolecular covalent attraction also influences the structure of the TTF·+ subunit,i.e.,its molecular plane is bent by an angle θ=5.6°.This work provides new knowledge on intermolecular interaction.

Enzyme Immobilization: An Overview on Methods, Support Material, and Applications of Immobilized Enzymes.

Science.gov (United States)

Sirisha, V L; Jain, Ankita; Jain, Amita

Immobilized enzymes can be used in a wide range of processes. In recent years, a variety of new approaches have emerged for the immobilization of enzymes that have greater efficiency and wider usage. During the course of the last two decades, this area has rapidly expanded into a multidisciplinary field. This current study is a comprehensive review of a variety of literature produced on the different enzymes that have been immobilized on various supporting materials. These immobilized enzymes have a wide range of applications. These include applications in the sugar, fish, and wine industries, where they are used for removing organic compounds from waste water. This study also reviews their use in sophisticated biosensors for metabolite control and in situ measurements of environmental pollutants. Immobilized enzymes also find significant application in drug metabolism, biodiesel and antibiotic production, bioremediation, and the food industry. The widespread usage of immobilized enzymes is largely due to the fact that they are cheaper, environment friendly, and much easier to use when compared to equivalent technologies. © 2016 Elsevier Inc. All rights reserved.

Assessing attitudes toward spinal immobilization.

Science.gov (United States)

Bouland, Andrew J; Jenkins, J Lee; Levy, Matthew J

2013-10-01

Prospective studies have improved knowledge of prehospital spinal immobilization. The opinion of Emergency Medical Services (EMS) providers regarding spinal immobilization is unknown, as is their knowledge of recent research advances. To examine the attitudes, knowledge, and comfort of prehospital and Emergency Department (ED) EMS providers regarding spinal immobilization performed under a non-selective protocol. An online survey was conducted from May to July of 2011. Participants were drawn from the Howard County Department of Fire and Rescue Services and the Howard County General Hospital ED. The survey included multiple choice questions and responses on a modified Likert scale. Correlation analysis and descriptive data were used to analyze results. Comfort using the Kendrick Extrication Device was low among ED providers. Experienced providers were more likely to indicate comfort using this device. Respondents often believed that spinal immobilization is appropriate in the management of penetrating trauma to the chest and abdomen. Reported use of padding decreased along with the frequency with which providers practice and encounter immobilized patients. Respondents often indicated that they perform spinal immobilization due solely to mechanism of injury. Providers who feel as if spinal immobilization is often performed unnecessarily were more likely to agree that immobilization causes an unnecessary delay in patient care. The results demonstrate the need for improved EMS education in the use of the Kendrick Extrication Device, backboard padding, and spinal immobilization in the management of penetrating trauma. The attitudes highlighted in this study are relevant to the implementation of a selective spinal immobilization protocol. Copyright © 2013 Elsevier Inc. All rights reserved.

The covalence effect of energy levels of ZnS:Mn2+

International Nuclear Information System (INIS)

Dong-Yang, Li; Mao-Lu, Du; Yi, Huang

2013-01-01

The contribution of the different covalence for t 2 and e orbitals must be considered in the investigation of the optical and magnetic properties of the transition metal ion in II–VI and III–V semiconductors. In present paper, two covalent parameters N t and N e associated with t 2 and e orbitals have been adopted to describe the covalence. The energy matrices considering the different covalence for t 2 and e orbitals have been provided for d 5 ions in crystal. These matrices show that the contribution from the Racah parameter A cannot be neglected in calculation of energy-level of d 5 ions in covalent crystal. The calculated results using the matrix show that the energy levels of 4 E and 4 A 1 states split, and the energy-level difference between 4 E and 4 A 1 states increases with increase of the different covalence between t 2 and e orbitals. These energy levels are always degenerate, when the different covalence for t 2 and e orbitals is neglected. By using the energy matrices, the energy-level of ZnS:Mn 2+ has been calculated. The calculated energy levels of ZnS:Mn 2+ are in good agreement with the experiments

Covalent functionalization of graphene with reactive intermediates.

Science.gov (United States)

Park, Jaehyeung; Yan, Mingdi

2013-01-15

Graphene, a material made exclusively of sp(2) carbon atoms with its π electrons delocalized over the entire 2D network, is somewhat chemically inert. Covalent functionalization can enhance graphene's properties including opening its band gap, tuning conductivity, and improving solubility and stability. Covalent functionalization of pristine graphene typically requires reactive species that can form covalent adducts with the sp(2) carbon structures in graphene. In this Account, we describe graphene functionalization reactions using reactive intermediates of radicals, nitrenes, carbenes, and arynes. These reactive species covalently modify graphene through free radical addition, CH insertion, or cycloaddition reactions. Free radical additions are among the most common reaction, and these radicals can be generated from diazonium salts and benzoyl peroxide. Electron transfer from graphene to aryl diazonium ion or photoactivation of benzoyl peroxide yields aryl radicals that subsequently add to graphene to form covalent adducts. Nitrenes, electron-deficient species generated by thermal or photochemical activation of organic azides, can functionalize graphene very efficiently. Because perfluorophenyl nitrenes show enhanced bimolecular reactions compared with alkyl or phenyl nitrenes, perfluorophenyl azides are especially effective. Carbenes are used less frequently than nitrenes, but they undergo CH insertion and C═C cycloaddition reactions with graphene. In addition, arynes can serve as a dienophile in a Diels-Alder type reaction with graphene. Further study is needed to understand and exploit the chemistry of graphene. The generation of highly reactive intermediates in these reactions leads to side products that complicate the product composition and analysis. Fundamental questions remain about the reactivity and regioselectivity of graphene. The differences in the basal plane and the undercoordinated edges of graphene and the zigzag versus arm-chair configurations

Immobilization of cholesterol esterase and cholesterol oxidase onto sol-gel films for application to cholesterol biosensor

Energy Technology Data Exchange (ETDEWEB)

Singh, Suman [Central Mechanical Engineering Research Institute, G. Avenue, Durgapur 713209, West Bengal (India); Singhal, Rahul [Biomolecular Electronics and Conducting Polymer Research Group, National Physical Laboratory, Dr. K.S. Krishnan Marg, New Delhi 110012 (India); Malhotra, B.D. [Biomolecular Electronics and Conducting Polymer Research Group, National Physical Laboratory, Dr. K.S. Krishnan Marg, New Delhi 110012 (India)]. E-mail: bansi.malhotra@gmail.com

2007-01-23

Cholesterol oxidase (ChOx) and cholesterol esterase (ChEt) have been covalently immobilized onto tetraethylorthosilicate (TEOS) sol-gel films. The tetraethylorthosilicate sol-gel/ChEt/ChOx enzyme films thus prepared have been characterized using scanning electron microscopic (SEM), UV-vis spectroscopic, Fourier-transform-infrared (FTIR) spectroscopic and amperometric techniques, respectively. The results of photometric measurements carried out on tetraethylorthosilicate sol-gel/ChEt/ChOx reveal thermal stability up to 55 deg. C, response time as 180 s, linearity up to 780 mg dL{sup -1} (12 mM), shelf life of 1 month, detection limit of 12 mg dL{sup -1} and sensitivity as 5.4 x 10{sup -5} Abs. mg{sup -1} dL{sup -1}.

Immobilization of cellulase by radiation polymerization

International Nuclear Information System (INIS)

Kumakura, M.; Kaetsu, I.

1983-01-01

Immobilization of cellulase by radiation polymerization at low temperatures was studied. The enzymatic activity of immobilized cellulase pellets varied with the monomer, enzyme concentration, and the thickness of immobilized cellulase pellets. The optimum monomer concentration in the immobilization of cellulase was 30-50% at the pellet thickness of 1.0 mm, in which the enzymatic activity was 50%. The enzymatic activity of immobilized cellulase pellets was examined using various substrates such as cellobiose, carboxymethylcellulose, and paper pretreated by radiation. It was found that irradiated paper can be hydrolyzed by immobilized cellulase pellets. (author)

Hydrogels Based on Dynamic Covalent and Non Covalent Bonds: A Chemistry Perspective

Directory of Open Access Journals (Sweden)

Francesco Picchioni

2018-03-01

Full Text Available Hydrogels based on reversible covalent bonds represent an attractive topic for research at both academic and industrial level. While the concept of reversible covalent bonds dates back a few decades, novel developments continue to appear in the general research area of gels and especially hydrogels. The reversible character of the bonds, when translated at the general level of the polymeric network, allows reversible interaction with substrates as well as responsiveness to variety of external stimuli (e.g., self-healing. These represent crucial characteristics in applications such as drug delivery and, more generally, in the biomedical world. Furthermore, the several possible choices that can be made in terms of reversible interactions generate an almost endless number of possibilities in terms of final product structure and properties. In the present work, we aim at reviewing the latest developments in this field (i.e., the last five years by focusing on the chemistry of the systems at hand. As such, this should allow molecular designers to develop a toolbox for the synthesis of new systems with tailored properties for a given application.

Quantitative Photochemical Immobilization of Biomolecules on Planar and Corrugated Substrates: A Versatile Strategy for Creating Functional Biointerfaces

Science.gov (United States)

Martin, Teresa A.; Herman, Christine T.; Limpoco, Francis T.; Michael, Madeline C.; Potts, Gregory K.; Bailey, Ryan C.

2014-01-01

Methods for the generation of substrates presenting biomolecules in a spatially controlled manner are enabling tools for applications in biosensor systems, microarray technologies, fundamental biological studies and biointerface science. We have implemented a method to create biomolecular patterns by using light to control the direct covalent immobilization of biomolecules onto benzophenone-modified glass substrates. We have generated substrates presenting up to three different biomolecules patterned in sequence, and demonstrate biomolecular photopatterning on corrugated substrates. The chemistry of the underlying monolayer was optimized to incorporate poly(ethylene glycol) to enable adhesive cell adhesion onto patterned extracellular matrix proteins. Substrates were characterized with contact angle goniometry, AFM, and immunofluorescence microscopy. Importantly, radioimmunoassays were performed to quantify the site density of immobilized biomolecules on photopatterned substrates. Retention of function of photopatterned proteins was demonstrated both by native ligand recognition and cell adhesion to photopatterned substrates, revealing that substrates generated with this method are suitable for probing specific cell receptor-ligand interactions. This molecularly general photochemical patterning method is an enabling tool that will allow the creation of substrates presenting both biochemical and topographical variation, which is an important feature of many native biointerfaces. PMID:21793535

Flexible strategy for immobilizing redox-active compounds using in situ generation of diazonium salts. Investigations of the blocking and catalytic properties of the layers.

Science.gov (United States)

Noël, Jean-Marc; Sjöberg, Béatrice; Marsac, Rémi; Zigah, Dodzi; Bergamini, Jean-François; Wang, Aifang; Rigaut, Stéphane; Hapiot, Philippe; Lagrost, Corinne

2009-11-03

A versatile two-step method is developed to covalently immobilize redox-active molecules onto carbon surfaces. First, a robust anchoring platform is grafted onto surfaces by electrochemical reduction of aryl diazonium salts in situ generated. Depending on the nature of the layer termini, -COOH or -NH(2), a further chemical coupling involving ferrocenemethylamine or ferrocene carboxylic acid derivatives leads to the covalent binding of ferrocene centers. The chemical strategy using acyl chloride activation is efficient and flexible, since it can be applied either to surface-reactive end groups or to reactive species in solution. Cyclic voltammetry analyses point to the covalent binding of ferrocene units restricted to the upper layers of the underlying aryl films, while AFM measurements show a lost of compactness of the layers after the chemical attachment of ferrocene centers. The preparation conditions of the anchoring layers were found to determine the interfacial properties of the resulted ferrocenyl-modified electrodes. The ferrocene units promoted effective redox mediation providing that the free redox probes are adequately chosen (i.e., vs size/formal potential) and the underlying layers exhibit strong blocking properties. For anchoring films with weaker blocking effect, the coexistence of two distinct phenomena, redox mediation and ET at pinholes could be evidenced.

Immobilized waste leaching

International Nuclear Information System (INIS)

Suarez, A.A.

1989-01-01

The main mechanism by which the immobilized radioactive materials can return to biosphere is the leaching due to the intrusion of water into the repositories. Some mathematical models and experiments utilized to evaluate the leaching rates in different immobilization matrices are described. (author) [pt

Plutonium Disposition by Immobilization

International Nuclear Information System (INIS)

Gould, T.; DiSabatino, A.; Mitchell, M.

2000-01-01

The ultimate goal of the Department of Energy (DOE) Immobilization Project is to develop, construct, and operate facilities that will immobilize between 17 to 50 tonnes (MT) of U.S. surplus weapons-usable plutonium materials in waste forms that meet the ''spent fuel'' standard and are acceptable for disposal in a geologic repository. Using the ceramic can-in-canister technology selected for immobilization, surplus plutonium materials will be chemically combined into ceramic forms which will be encapsulated within large canisters of high level waste (HLW) glass. Deployment of the immobilization capability should occur by 2008 and be completed within 10 years. In support of this goal, the DOE Office of Fissile Materials Disposition (MD) is conducting development and testing (D and T) activities at four DOE laboratories under the technical leadership of Lawrence Livermore National Laboratory (LLNL). The Savannah River Site has been selected as the site for the planned Plutonium Immobilization Plant (PIP). The D and T effort, now in its third year, will establish the technical bases for the design, construction, and operation of the U. S. capability to immobilize surplus plutonium in a suitable and cost-effective manner. Based on the D and T effort and on the development of a conceptual design of the PIP, automation is expected to play a key role in the design and operation of the Immobilization Plant. Automation and remote handling are needed to achieve required dose reduction and to enhance operational efficiency

Binary polypeptide system for permanent and oriented protein immobilization

Directory of Open Access Journals (Sweden)

Bailes Julian

2010-05-01

Full Text Available Abstract Background Many techniques in molecular biology, clinical diagnostics and biotechnology rely on binary affinity tags. The existing tags are based on either small molecules (e.g., biotin/streptavidin or glutathione/GST or peptide tags (FLAG, Myc, HA, Strep-tag and His-tag. Among these, the biotin-streptavidin system is most popular due to the nearly irreversible interaction of biotin with the tetrameric protein, streptavidin. The major drawback of the stable biotin-streptavidin system, however, is that neither of the two tags can be added to a protein of interest via recombinant means (except for the Strep-tag case leading to the requirement for chemical coupling. Results Here we report a new immobilization system which utilizes two monomeric polypeptides which self-assemble to produce non-covalent yet nearly irreversible complex which is stable in strong detergents, chaotropic agents, as well as in acids and alkali. Our system is based on the core region of the tetra-helical bundle known as the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex. This irreversible protein attachment system (IPAS uses either a shortened syntaxin helix and fused SNAP25-synaptobrevin or a fused syntaxin-synaptobrevin and SNAP25 allowing a two-component system suitable for recombinant protein tagging, capture and immobilization. We also show that IPAS is suitable for use with traditional beads and chromatography, planar surfaces and Biacore, gold nanoparticles and for protein-protein interaction in solution. Conclusions IPAS offers an alternative to chemical cross-linking, streptavidin-biotin system and to traditional peptide affinity tags and can be used for a wide range of applications in nanotechnology and molecular sciences.

Membranes suited for immobilizing biomolecules

NARCIS (Netherlands)

2009-01-01

The present invention relates to flow-through membranes suitable for the immobilization of biomols., methods for the prepn. of such membranes and the use of such membranes for the immobilization of biomols. and subsequent detection of immobilized biomols. The invention concerns a flow-through

Immobilization of Platelet-Rich Plasma onto COOH Plasma-Coated PCL Nanofibers Boost Viability and Proliferation of Human Mesenchymal Stem Cells

Directory of Open Access Journals (Sweden)

Anastasiya Solovieva

2017-12-01

Full Text Available The scaffolds made of polycaprolactone (PCL are actively employed in different areas of biology and medicine, especially in tissue engineering. However, the usage of unmodified PCL is significantly restricted by the hydrophobicity of its surface, due to the fact that its inert surface hinders the adhesion of cells and the cell interactions on PCL surface. In this work, the surface of PCL nanofibers is modified by Ar/CO2/C2H4 plasma depositing active COOH groups in the amount of 0.57 at % that were later used for the immobilization of platelet-rich plasma (PRP. The modification of PCL nanofibers significantly enhances the viability and proliferation (by hundred times of human mesenchymal stem cells, and decreases apoptotic cell death to a normal level. According to X-ray photoelectron spectroscopy (XPS, after immobilization of PRP, up to 10.7 at % of nitrogen was incorporated into the nanofibers surface confirming the grafting of proteins. Active proliferation and sustaining the cell viability on nanofibers with immobilized PRP led to an average number of cells of 258 ± 12.9 and 364 ± 34.5 for nanofibers with ionic and covalent bonding of PRP, respectively. Hence, our new method for the modification of PCL nanofibers with PRP opens new possibilities for its application in tissue engineering.

Immobilized enzymes and cells

Energy Technology Data Exchange (ETDEWEB)

Bucke, C; Wiseman, A

1981-04-04

This article reviews the current state of the art of enzyme and cell immobilization and suggests advances which might be made during the 1980's. Current uses of immobilized enzymes include the use of glucoamylase in the production of glucose syrups from starch and glucose isomerase in the production of high fructose corn syrup. Possibilities for future uses of immobilized enzymes and cells include the utilization of whey and the production of ethanol.

Immobilized copper(II) macrocyclic complex on MWCNTs with antibacterial activity

Energy Technology Data Exchange (ETDEWEB)

Tarlani, Aliakbar, E-mail: Tarlani@ccerci.ac.ir [Inorganic Nanostructures and Catalysts Research Lab., Chemistry & Chemical Engineering Research Center of Iran, Pajoohesh Blvd., km 17, Karaj Hwy, Tehran 14968-13151 (Iran, Islamic Republic of); Narimani, Khashayar [Inorganic Nanostructures and Catalysts Research Lab., Chemistry & Chemical Engineering Research Center of Iran, Pajoohesh Blvd., km 17, Karaj Hwy, Tehran 14968-13151 (Iran, Islamic Republic of); Mohammadipanah, Fatemeh; Hamedi, Javad [Department of Microbial Biotechnology, School of Biology and Center of Excellence in Phylogeny of Living Organisms, College of Science, University of Tehran, Tehran 14155-6455 (Iran, Islamic Republic of); University of Tehran Biocompound Collection (UTBC), Microbial Technology and Products Research Center, University of Tehran, Tehran (Iran, Islamic Republic of); Tahermansouri, Hasan [Department of Chemistry, Ayatollah Amoli Branch, Islamic Azad University, Amol (Iran, Islamic Republic of); Amini, Mostafa M. [Department of Chemistry, Shahid Behshti University, 1983963113, Tehran (Iran, Islamic Republic of)

2015-06-30

Graphical abstract: In an antibacterial test, grafted copper(II) macrocyclic complex on the surface of MWCNT showed higher antibacterial activity against Bacillus subtilis compared to the individual MWCNT-COOH and the complex. - Highlights: • Copper(II) tetraaza macrocyclic complex covalently bonded to modified MWCNT. • Grafting of the complex carried out via an interaction between −C(=O)Cl group and NH of the ligand. • The samples were subjected in an antibacterial assessment to compare their activity. • Immobilized complex showed higher antibacterial activity against Bacillus subtilis ATCC 6633 compared to separately MWCNT-C(C=O)-OH and CuTAM. - Abstract: In a new approach, a copper(II) tetraaza macrocyclic complex (CuTAM) was covalently bonded on modified multi-walled carbon nanotubes (MWCNTs). To achieve this purpose, MWCNTs were converted to MWCNT-COCl and then reacted to NH groups of TAM ligand. The prepared material was characterized by Fourier Transform Infrared (FT-IR), X-ray diffraction (XRD), Raman spectroscopy, thermal gravimetric analysis (TGA), and FESEM (field emission scanning electron microscopy). FT-IR and TGA demonstrated the presence of the organic moieties, and XRD proved that the structure of MWCNTs remained intact during the three modification steps. An increase in the I{sub D}/I{sub G} ratio in Raman spectra confirmed the surface modifications. Finally, the samples were subjected to an antibacterial assessment to compare their biological activity. The antibacterial test showed that the grafted complex on the surface of the nanotube (MWCNT-CO-CuTAM) has higher antibacterial activity against Bacillus subtilis ATCC 6633 than the MWCNT-COOH and CuTAM with 1000 and 2000 μg/mL.

Hybrid Organometallic-Inorganic Nanomaterial: Acetyl Ferrocene Schiff base Immobilized on Silica Coated Magnetite Nanoparticles

Directory of Open Access Journals (Sweden)

M. Masteri-Farahani

2015-10-01

Full Text Available In  this  work,  a  new  hybrid  organometallic-inorganic  hybrid nanomaterial was prepared by immobilization of acetyl ferrocene on the  surface  of magnetite  nanoparticles. Covalent  grafting of silica coated magnetite nanoparticles (SCMNPs with 3-aminopropyl triethoxysilane gave aminopropyl-modified magnetite nanoparticles (AmpSCMNPs. Then, Schiff base condensation  of AmpSCMNPs with acetyl  ferrocene resulted in the preparation of acferro-SCMNPs hybrid nanomaterial. Characterization of the prepared nanomaterial was performed with different physicochemical methods such as Fourier transform infrared spectroscopy (FT-IR, X-ray diffraction (XRD, vibrating sample magnetometry (VSM, thermogravimetric analysis (TGA, scanning electron microscopy (SEM, and transmission electron microscopy (TEM. VSM analysis showed superparamagnetic properties of the prepared nanomaterial and TEM and SEM analyses indicated the relatively spherical nanoparticles with 15 nm average size.

Microorganism immobilization

Science.gov (United States)

Compere, Alicia L.; Griffith, William L.

1981-01-01

Live metabolically active microorganisms are immobilized on a solid support by contacting particles of aggregate material with a water dispersible polyelectrolyte such as gelatin, crosslinking the polyelectrolyte by reacting it with a crosslinking agent such as glutaraldehyde to provide a crosslinked coating on the particles of aggregate material, contacting the coated particles with live microorganisms and incubating the microorganisms in contact with the crosslinked coating to provide a coating of metabolically active microorganisms. The immobilized microorganisms have continued growth and reproduction functions.

Electrocatalytic oxidation of ascorbic acid using a single layer of gold nanoparticles immobilized on 1,6-hexanedithiol modified gold electrode

International Nuclear Information System (INIS)

Sivanesan, A.; Kannan, P.; Abraham John, S.

2007-01-01

This paper describes the electrocatalytic oxidation of ascorbic acid (AA) in phosphate buffer solution by the immobilized citrate capped gold nanoparticles (AuNPs) on 1,6-hexanedithiol (HDT) modified Au electrode. X-ray photoelectron spectrum (XPS) of HDT suggests that it forms a monolayer on Au surface through one of the two -SH groups and the other -SH group is pointing away from the electrode surface. The free -SH groups of HDT were used to covalently attach colloidal AuNPs. The covalent attachment of AuNPs on HDT monolayer was confirmed from the observed characteristic carboxylate ion stretching modes of citrate attached with AuNPs in the infra-red reflection absorption spectrum (IRRAS) in addition to a higher reductive desorption charges obtained for AuNPs immobilized on HDT modified Au (Au/HDT/AuNPs) electrode in 0.1 M KOH when compared to HDT modified Au (Au/HDT) electrode. The electron transfer reaction of [Fe(CN) 6 ] 4-/3- was markedly hindered at the HDT modified Au (Au/HDT) electrode while it was restored with a peak separation of 74 mV after the immobilization of AuNPs on Au/HDT (Au/HDT/AuNPs) electrode indicating a good electronic communication between the immobilized AuNPs and the underlying bulk Au electrode through a HDT monolayer. The Cottrell slope obtained from the potential-step chronoamperometric measurements for the reduction of ferricyanide at Au/HDT/AuNPs was higher than that of bare Au electrode indicating the increased effective surface area of AuNPs modified electrode. The Au/HDT/AuNPs electrode exhibits excellent electrocatalytic activity towards the oxidation of ascorbic acid (AA) by enhancing the oxidation peak current to more than two times with a 210 mV negative shift in the oxidation potential when compared to a bare Au electrode. The standard heterogeneous electron transfer rate constant (k s ) calculated for AA oxidation at Au/HDT/AuNPs electrode was 5.4 x 10 -3 cm s -1 . The oxidation peak of AA at Au/HDT/AuNPs electrode was

Immobilization of Lipase from Penicillium sp. Section Gracilenta (CBMAI 1583) on Different Hydrophobic Supports: Modulation of Functional Properties.

Science.gov (United States)

Turati, Daniela F M; Morais Júnior, Wilson G; Terrasan, César R F; Moreno-Perez, Sonia; Pessela, Benevides C; Fernandez-Lorente, Gloria; Guisan, Jose M; Carmona, Eleonora C

2017-02-22

Lipases are promising enzymes that catalyze the hydrolysis of triacylglycerol ester bonds at the oil/water interface. Apart from allowing biocatalyst reuse, immobilization can also affect enzyme structure consequently influencing its activity, selectivity, and stability. The lipase from Penicillium sp. section Gracilenta (CBMAI 1583) was successfully immobilized on supports bearing butyl, phenyl, octyl, octadecyl, and divinylbenzyl hydrophobic moieties wherein lipases were adsorbed through the highly hydrophobic opened active site. The highest activity in aqueous medium was observed for the enzyme adsorbed on octyl support, with a 150% hyperactivation regarding the soluble enzyme activity, and the highest adsorption strength was verified with the most hydrophobic support (octadecyl Sepabeads), requiring 5% Triton X-100 to desorb the enzyme from the support. Most of the derivatives presented improved properties such as higher stability to pH, temperature, and organic solvents than the covalently immobilized CNBr derivative (prepared under very mild experimental conditions and thus a reference mimicking free-enzyme behavior). A 30.8- and 46.3-fold thermostabilization was achieved in aqueous medium, respectively, by the octyl Sepharose and Toyopearl butyl derivatives at 60 °C, in relation to the CNBr derivative. The octyl- and phenyl-agarose derivatives retained 50% activity after four and seven cycles of p -nitrophenyl palmitate hydrolysis, respectively. Different derivatives exhibited different properties regarding their properties for fish oil hydrolysis in aqueous medium and ethanolysis in anhydrous medium. The most active derivative in ethanolysis of fish oil was the enzyme adsorbed on a surface covered by divinylbenzyl moieties and it was 50-fold more active than the enzyme adsorbed on octadecyl support. Despite having identical mechanisms of immobilization, different hydrophobic supports seem to promote different shapes of the adsorbed open active site of the

Supramolecular protein immobilization on lipid bilayers

NARCIS (Netherlands)

Bosmans, R.P.G.; Hendriksen, W.E.; Verheijden, Mark Lloyd; Eelkema, R.; Jonkheijm, Pascal; van Esch, J.H.; Brunsveld, Luc

2015-01-01

Protein immobilization on surfaces, and on lipid bilayers specifically, has great potential in biomolecular and biotechnological research. Of current special interest is the immobilization of proteins using supramolecular noncovalent interactions. This allows for a reversible immobilization and

Non-Covalent Supported of l-Proline on Graphene Oxide/Fe3O4 Nanocomposite: A Novel, Highly Efficient and Superparamagnetically Separable Catalyst for the Synthesis of Bis-Pyrazole Derivatives

Directory of Open Access Journals (Sweden)

Mosadegh Keshavarz

2018-02-01

Full Text Available A superparamagnetic graphene oxide/Fe3O4/l-proline nano hybrid that was obtained from the non-covalent immobilization of l-proline on graphene oxide/Fe3O4 nanocomposite was used as a new magnetically separable catalyst for the efficient synthesis of 4,4′-(arylmethylenebis(1H-pyrazol-5-ol derivatives. The prepared heterogeneous catalyst was characterized using FTIR, TGA, DTG, XRD, TEM, SEM, and elemental analysis techniques. Short reaction times (5–15 min, excellent yields (87–98%, and simple experimental procedure with an easy work-up are some of the advantages of the introduced catalyst.

Patterned immobilization of antibodies within roll-to-roll hot embossed polymeric microfluidic channels.

Directory of Open Access Journals (Sweden)

Belachew Feyssa

Full Text Available This paper describes a method for the patterned immobilization of capture antibodies into a microfluidic platform fabricated by roll-to-roll (R2R hot embossing on poly (methyl methacrylate (PMMA. Covalent attachment of antibodies was achieved by two sequential inkjet printing steps. First, a polyethyleneimine (PEI layer was deposited onto oxygen plasma activated PMMA foil and further cross-linked with glutaraldehyde (GA to provide an amine-reactive aldehyde surface (PEI-GA. This step was followed by a second deposition of antibody by overprinting on the PEI-GA patterned PMMA foil. The PEI polymer ink was first formulated to ensure stable drop formation in inkjet printing and the printed films were characterized using atomic force microscopy (AFM and X-ray photoelectron spectroscopy (XPS. Anti-CRP antibody was patterned on PMMA foil by the developed method and bonded permanently with R2R hot embossed PMMA microchannels by solvent bonding lamination. The functionality of the immobilized antibody inside the microfluidic channel was evaluated by fluorescence-based sandwich immunoassay for detection of C-reactive protein (CRP. The antibody-antigen assay exhibited a good level of linearity over the range of 10 ng/ml to 500 ng/ml (R(2 = 0.991 with a calculated detection limit of 5.2 ng/ml. The developed patterning method is straightforward, rapid and provides a versatile approach for creating multiple protein patterns in a single microfluidic channel for multiplexed immunoassays.

An inexpensive and simple method for thermally stable immobilization of DNA on an unmodified glass surface: UV linking of poly(T)10-poly(C)10-tagged DNA probes

DEFF Research Database (Denmark)

Guðnason, Haukur; Dufva, Hans Martin; Bang, Dang Duong

2008-01-01

be linked by UV light irradiation onto a plain, unmodified glass surface. Probes immobilized onto unmodified glass microscope slides performed similarly to probes bound to commercial amino-silane-coated slides and had comparable detection limits. The TC-tagged probes linked to unmodified glass did not show...... any significant decrease in hybridization performance after a 20 min incubation in water at 100 degrees C prior to rehybridization, indicating a covalent bond between the TC tag and unmodified glass. The probes were used in thermal minisequencing cycling reactions. Furthermore, the TC tag improved...

Immobilization of enzymes by radiation

International Nuclear Information System (INIS)

Kaetsu, I.; Kumakura, M.; Yoshida, M.; Asano, M.; Himei, M.; Tamura, M.; Hayashi, K.

1979-01-01

Immobilization of various enzymes was performed by radiation-induced polymerization of glass-forming monomers at low temperatures. Alpha-amylase and glucoamylase were effectively immobilized in hydrophilic polymer carrier such as poly(2-hydroxyethyl methacrylate) and also in rather hydrophobic carrier such as poly(tetraethylene-glycol diacrylate). Immobilized human hemoglobin underwent the reversible oxygenation concomitantly with change of oxygen concentration outside of the matrices. (author)

The Synthesis, Structures and Chemical Properties of Macrocyclic Ligands Covalently Bonded into Layered Arrays

International Nuclear Information System (INIS)

Clearfield, Abraham

2003-01-01

OAK-B135 The immobilization of crown ethers tends to limit the leveling effect of solvents making the macrocycles more selective. In addition immobilization has the added advantage of relative ease of recovery of the otherwise soluble crown. We have affixed CH2PO3H2 groups to azacrown ethers. The resultant phosphorylated macrocycles may spontaneously aggregate into crystalline supramolecular linear arrays or contacted with cations produce layered or linear polymers. In the linear polymers the metal and phosphonic acids covalently bond into a central stem with the macrocyclic rings protruding from the stem as leaves on a twig. Two types of layered compounds were obtained with group 4 metals. Monoaza-crown ethers form a bilayer where the M4+ plus phosphonic acid groups build the layer and the rings fill the interlayer space. 1, 10-diazadiphosphonic acids cross-link the metal phosphonate layers forming a three-dimensional array of crown ethers. In order to improve diffusion into these 3-D arrays they are spaced by inclusion of phosphate or phosphate groups. Two series of azamacrocylic crown ethers were prepared containing rings with 20 to 32 atoms. These larger rings can complex two cations per ring. Methylene phosphonic acid groups have been bonded to the aza ring atoms to increase the complexing ability of these ligands. Our approach is to carry out acid-base titrations in the absence and presence of cations to determine the pKa values of the protons, both those bonded to aza groups and those associated with the phosphonic acid groups. From the differences in the titration curves obtained with and without the cations present we obtain the stoichiometry of complex formation and the complex stability constants. Some of the applications we are targeting include phase transfer catalysis, separation of cations and the separation of radioisotopes for diagnostic and cancer therapeutic purposes

Covalent immobilization of stem cell factor and stromal derived factor 1α for in vitro culture of hematopoietic progenitor cells.

Science.gov (United States)

Cuchiara, Maude L; Horter, Kelsey L; Banda, Omar A; West, Jennifer L

2013-12-01

Hematopoietic stem cells (HSCs) are currently utilized in the treatment of blood diseases, but widespread application of HSC therapeutics has been hindered by the limited availability of HSCs. With a better understanding of the HSC microenvironment and the ability to precisely recapitulate its components, we may be able to gain control of HSC behavior. In this work we developed a novel, biomimetic PEG hydrogel material as a substrate for this purpose and tested its potential with an anchorage-independent hematopoietic cell line, 32D clone 3 cells. We immobilized a fibronectin-derived adhesive peptide sequence, RGDS; a cytokine critical in HSC self-renewal, stem cell factor (SCF); and a chemokine important in HSC homing and lodging, stromal derived factor 1α (SDF1α), onto the surfaces of poly(ethylene glycol) (PEG) hydrogels. To evaluate the system's capabilities, we observed the effects of the biomolecules on 32D cell adhesion and morphology. We demonstrated that the incorporation of RGDS onto the surfaces promotes 32D cell adhesion in a dose-dependent fashion. We also observed an additive response in adhesion on surfaces with RGDS in combination with either SCF or SDF1α. In addition, the average cell area increased and circularity decreased on gel surfaces containing immobilized SCF or SDF1α, indicating enhanced cell spreading. By recapitulating aspects of the HSC microenvironment using a PEG hydrogel scaffold, we have shown the ability to control the adhesion and spreading of the 32D cells and demonstrated the potential of the system for the culture of primary hematopoietic cell populations. Copyright © 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Analysis of the surface density and reactivity of perfluorophenylazide and the impact on ligand immobilization

Energy Technology Data Exchange (ETDEWEB)

Zorn, Gilad, E-mail: zorn@ge.com; Castner, David G. [National ESCA and Surface Analysis Center for Biomedical Problems, Departments of Bioengineering and Chemical Engineering, University of Washington, Box 351653, Seattle, Washington 98195-1653 (United States); Tyagi, Anuradha; Wang, Xin; Wang, Hui; Yan, Mingdi, E-mail: Mingdi-Yan@uml.edu [Department of Chemistry, Portland State University, Portland, Oregon 97207-0751 (United States)

2015-03-15

Perfluorophenylazide (PFPA) chemistry is a novel method for tailoring the surface properties of solid surfaces and nanoparticles. It is general and versatile, and has proven to be an efficient way to immobilize graphene, proteins, carbohydrates, and synthetic polymers. The main thrust of this work is to provide a detailed investigation on the chemical composition and surface density of the PFPA tailored surface. Specifically, gold surfaces were treated with PFPA-derivatized (11-mercaptoundecyl)tetra(ethylene glycol) (PFPA-MUTEG) mixed with 2-[2-(2-mercaptoethoxy)ethoxy]ethanol (MDEG) at varying solution mole ratios. Complementary analytical techniques were employed to characterize the resulting films including Fourier transform infrared spectroscopy to detect fingerprints of the PFPA group, x-ray photoelectron spectroscopy and ellipsometry to study the homogeneity and uniformity of the films, and near edge x-ray absorption fine structures to study the electronic and chemical structure of the PFPA groups. Results from these studies show that the films prepared from 90:10 and 80:20 PFPA-MUTEG/MDEG mixed solutions exhibited the highest surface density of PFPA and the most homogeneous coverage on the surface. A functional assay using surface plasmon resonance with carbohydrates covalently immobilized onto the PFPA-modified surfaces showed the highest binding affinity for lectin on the PFPA-MUTEG/MDEG film prepared from a 90:10 solution.

Immobilized fluid membranes for gas separation

Science.gov (United States)

Liu, Wei; Canfield, Nathan L; Zhang, Jian; Li, Xiaohong Shari; Zhang, Jiguang

2014-03-18

Provided herein are immobilized liquid membranes for gas separation, methods of preparing such membranes and uses thereof. In one example, the immobilized membrane includes a porous metallic host matrix and an immobilized liquid fluid (such as a silicone oil) that is immobilized within one or more pores included within the porous metallic host matrix. The immobilized liquid membrane is capable of selective permeation of one type of molecule (such as oxygen) over another type of molecule (such as water). In some examples, the selective membrane is incorporated into a device to supply oxygen from ambient air to the device for electrochemical reactions, and at the same time, to block water penetration and electrolyte loss from the device.

Coval: improving alignment quality and variant calling accuracy for next-generation sequencing data.

Directory of Open Access Journals (Sweden)

Shunichi Kosugi

Full Text Available Accurate identification of DNA polymorphisms using next-generation sequencing technology is challenging because of a high rate of sequencing error and incorrect mapping of reads to reference genomes. Currently available short read aligners and DNA variant callers suffer from these problems. We developed the Coval software to improve the quality of short read alignments. Coval is designed to minimize the incidence of spurious alignment of short reads, by filtering mismatched reads that remained in alignments after local realignment and error correction of mismatched reads. The error correction is executed based on the base quality and allele frequency at the non-reference positions for an individual or pooled sample. We demonstrated the utility of Coval by applying it to simulated genomes and experimentally obtained short-read data of rice, nematode, and mouse. Moreover, we found an unexpectedly large number of incorrectly mapped reads in 'targeted' alignments, where the whole genome sequencing reads had been aligned to a local genomic segment, and showed that Coval effectively eliminated such spurious alignments. We conclude that Coval significantly improves the quality of short-read sequence alignments, thereby increasing the calling accuracy of currently available tools for SNP and indel identification. Coval is available at http://sourceforge.net/projects/coval105/.

Effects of immobilization on spermiogenesis

Science.gov (United States)

Meitner, E. R.

1980-01-01

The influence of immobilization stress on spermiogenesis in rats was investigated. After 96 hour immobilization, histological changes began to manifest themselves in the form of practically complete disappearance of cell population of the wall of seminiferous tubule as well as a markedly increased number of cells with pathologic mitoses. Enzymological investigations showed various changes of activity (of acid and alkaline phosphatase and nonspecific esterase) in the 24, 48, and 96 hour immobilization groups.

Surface cell immobilization within perfluoroalkoxy microchannels

Energy Technology Data Exchange (ETDEWEB)

Stojkovič, Gorazd; Krivec, Matic [Faculty of Chemistry and Chemical Technology, University of Ljubljana, Aškerčeva 5, SI-1000 Ljubljana (Slovenia); Vesel, Alenka [Jožef Stefan Institute, Jamova cesta 39, 1000 Ljubljana (Slovenia); Marinšek, Marjan [Faculty of Chemistry and Chemical Technology, University of Ljubljana, Aškerčeva 5, SI-1000 Ljubljana (Slovenia); Žnidaršič-Plazl, Polona, E-mail: polona.znidarsic@fkkt.uni-lj.si [Faculty of Chemistry and Chemical Technology, University of Ljubljana, Aškerčeva 5, SI-1000 Ljubljana (Slovenia)

2014-11-30

Graphical abstract: - Highlights: • A very efficient approach for immobilization of cells into microreactors is presented. • It is applicable to various materials, including PFA and cyclic olefin (co)polymers. • It was used to immobilize different prokaryotic and eukaryotic microbes. • Cells were immobilized on the surface in high density and showed good stability. • Mechanisms of APTES interactions with target materials are proposed. - Abstract: Perfluoroalkoxy (PFA) is one of the most promising materials for the fabrication of cheap, solvent resistant and reusable microfluidic chips, which have been recently recognized as effective tools for biocatalytic process development. The application of biocatalysts significantly depends on efficient immobilization of enzymes or cells within the reactor enabling long-term biocatalyst use. Functionalization of PFA microchannels by 3-aminopropyltriethoxysilane (ATPES) and glutaraldehyde was used for rapid preparation of microbioreactors with surface-immobilized cells. X-ray photoelectron spectroscopy and scanning electron microscopy were used to accurately monitor individual treatment steps and to select conditions for cell immobilization. The optimized protocol for Saccharomyces cerevisiae immobilization on PFA microchannel walls comprised ethanol surface pretreatment, 4 h contacting with 10% APTES aqueous solution, 10 min treatment with 1% glutaraldehyde and 20 min contacting with cells in deionized water. The same protocol enabled also immobilization of Escherichia coli, Pseudomonas putida and Bacillus subtilis cells on PFA surface in high densities. Furthermore, the developed procedure has been proved to be very efficient also for surface immobilization of tested cells on other materials that are used for microreactor fabrication, including glass, polystyrene, poly (methyl methacrylate), polycarbonate, and two olefin-based polymers, namely Zeonor{sup ®} and Topas{sup ®}.

Capillary electrophoresis of covalently functionalized single-chirality carbon nanotubes.

Science.gov (United States)

He, Pingli; Meany, Brendan; Wang, Chunyan; Piao, Yanmei; Kwon, Hyejin; Deng, Shunliu; Wang, YuHuang

2017-07-01

We demonstrate the separation of chirality-enriched single-walled carbon nanotubes (SWCNTs) by degree of surface functionalization using high-performance CE. Controlled amounts of negatively charged and positively charged functional groups were attached to the sidewall of chirality-enriched SWCNTs through covalent functionalization using 4-carboxybenzenediazonium tetrafluoroborate or 4-diazo-N,N-diethylaniline tetrafluoroborate, respectively. Surfactant- and pH-dependent studies confirmed that under conditions that minimized ionic screening effects, separation of these functionalized SWCNTs was strongly dependent on the surface charge density introduced through covalent surface chemistry. For both heterogeneous mixtures and single-chirality-enriched samples, covalently functionalized SWCNTs showed substantially increased peak width in electropherogram spectra compared to nonfunctionalized SWCNTs, which can be attributed to a distribution of surface charges along the functionalized nanotubes. Successful separation of functionalized single-chirality SWCNTs by functional density was confirmed with UV-Vis-NIR absorption and Raman scattering spectroscopies of fraction collected samples. These results suggest a high degree of structural heterogeneity in covalently functionalized SWCNTs, even for chirality-enriched samples, and show the feasibility of applying CE for high-performance separation of nanomaterials based on differences in surface functional density. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Remote handling in the Plutonium Immobilization Project: Plutonium conversion and first stage immobilization

International Nuclear Information System (INIS)

Brault, J.R.

2000-01-01

Since the break up of the Soviet Union at the end of the Cold War, the United States and Russia have been negotiating ways to reduce their nuclear stockpiles. Economics is one of the reasons behind this, but another important reason is safeguarding these materials from unstable organizations and countries. With the downsizing of the nuclear stockpiles, large quantities of plutonium are being declared excess and must be safely disposed of. The Savannah River Site (SRS) has been selected as the site where the immobilization facility will be located. Conceptual design and process development commenced in 1998. SRS will immobilize excess plutonium in a ceramic waste form and encapsulate it in vitrified high level waste in the Defense Waste Processing Facility (DWPF) canister. These canisters will then be interred in the national repository at Yucca Mountain, New Mexico. The facility is divided into three distinct operating areas: Plutonium Conversion, First Stage Immobilization, and Second Stage Immobilization. This paper will discuss the first two operations

Covalent and non-covalent chemical engineering of actin for biotechnological applications.

Science.gov (United States)

Kumar, Saroj; Mansson, Alf

2017-11-15

The cytoskeletal filaments are self-assembled protein polymers with 8-25nm diameters and up to several tens of micrometres length. They have a range of pivotal roles in eukaryotic cells, including transportation of intracellular cargoes (primarily microtubules with dynein and kinesin motors) and cell motility (primarily actin and myosin) where muscle contraction is one example. For two decades, the cytoskeletal filaments and their associated motor systems have been explored for nanotechnological applications including miniaturized sensor systems and lab-on-a-chip devices. Several developments have also revolved around possible exploitation of the filaments alone without their motor partners. Efforts to use the cytoskeletal filaments for applications often require chemical or genetic engineering of the filaments such as specific conjugation with fluorophores, antibodies, oligonucleotides or various macromolecular complexes e.g. nanoparticles. Similar conjugation methods are also instrumental for a range of fundamental biophysical studies. Here we review methods for non-covalent and covalent chemical modifications of actin filaments with focus on critical advantages and challenges of different methods as well as critical steps in the conjugation procedures. We also review potential uses of the engineered actin filaments in nanotechnological applications and in some key fundamental studies of actin and myosin function. Finally, we consider possible future lines of investigation that may be addressed by applying chemical conjugation of actin in new ways. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Covalently linked bisporphyrins bearing tetraphenylporphyrin and ...

Indian Academy of Sciences (India)

Covalently linked bisporphyrins bearing tetraphenylporphyrin and perbromoporphyrin units: Synthesis and their properties. Puttaiah Bhyrappa V Krishnan ... yields of the TPP moiety. Electrochemical redox and fluorescence data seem to suggest the possible existence of intramolecular interactions in these bisporphyrins.

Immobilized cells of Candida rugosa possessing fumarase activity

Energy Technology Data Exchange (ETDEWEB)

Yang, L.; Zhone, L.

1980-01-01

Immobilized cells of C. rugosa that possessed fumarase activity were prepared by different methods; the most active immobilized cells were entrapped in polyacrylamide gels. The effects of pH temperature, and divalent cations on the fumarase activity of both immobilized and native cells were the same. Mn/sup 2 +/, Mg/sup 2 +/, Ca/sup 2 +/, and Fe/sup 2 +/ did not protect the immobilized enzyme against thermal inactivation. The activity of immobilized fumarase remained constant during 91 days of storage of 4-6 degrees. The immobilized cell column was used for the continuous production of L-malic acid from 1M fumarate at 30 degrees and pH 8.5. The immobilized column operated steadily for 2 months. Half life of the immobilized fumarase at 30 degrees was 95 days.

Covalent Coupling of Organophosphorus Hydrolase Loaded Quantum Dots to Carbon Nanotube/Au Nanocomposite for Enhanced Detection of Methyl Parathion

Energy Technology Data Exchange (ETDEWEB)

Du, Dan; Chen, Wenjuan; Zhang, Weiying; Liu, Deli; Li, Haibing; Lin, Yuehe

2010-02-15

An amperometric biosensor for highly selective and sensitive determination of methyl parathion (MP) was developed based on dual signal amplification: (1) a large amount of introduced enzyme on the electrode surface and (2) synergistic effects of nanoparticles towards enzymatic catalysis. The fabrication process includes (1) electrochemical deposition of gold nanoparticles by a multi-potential step technique at multiwalled carbon nanotube (MWCNT) film pre-cast on a glassy carbon electrode and (2) immobilization of methyl parathion degrading enzyme (MPDE) onto a modified electrode through CdTe quantum dots (CdTe QDs) covalent attachment. The introduced MWCNT and gold nanoparticles significantly increased the surface area and exhibited synergistic effects towards enzymatic catalysis. CdTe QDs are further used as carriers to load a large amount of enzyme. As a result of these two important enhancement factors, the proposed biosensor exhibited extremely sensitive, perfectly selective, and rapid response to methyl parathion in the absence of a mediator.

Immobilization needs and technology programs

International Nuclear Information System (INIS)

Gray, L.W.; Kan, T.; Shaw, H.; Armantrout, G.

1995-01-01

In the aftermath of the Cold War, the US and Russia agreed to large reductions in nuclear weapons. To aid in the selection of long-term management options, DOE has undertaken a multifaceted study to select options for storage and disposition of plutonium in keeping with US policy that plutonium must be subjected to the highest standards of safety, security, and accountability. One alternative being considered is immobilization. To arrive at a suitable immobilization form, we first reviewed published information on high-level waste immobilization technologies and identified 72 possible plutonium immobilization forms to be prescreened. Surviving forms were further screened using multi-attribute utility analysis to determine the most promising technology families. Promising immobilization families were further evaluated to identify chemical, engineering, environmental, safety, and health problems that remain to be solved prior to making technical decisions as to the viability of using the form for long- term disposition of plutonium. From this evaluation, a detailed research and development plan has been developed to provide answers to these remaining questions

Biodiesel production with immobilized lipase: A review.

Science.gov (United States)

Tan, Tianwei; Lu, Jike; Nie, Kaili; Deng, Li; Wang, Fang

2010-01-01

Fatty acid alkyl esters, also called biodiesel, are environmentally friendly and show great potential as an alternative liquid fuel. Biodiesel is produced by transesterification of oils or fats with chemical catalysts or lipase. Immobilized lipase as the biocatalyst draws high attention because that process is "greener". This article reviews the current status of biodiesel production with immobilized lipase, including various lipases, immobilization methods, various feedstocks, lipase inactivation caused by short chain alcohols and large scale industrialization. Adsorption is still the most widely employed method for lipase immobilization. There are two kinds of lipase used most frequently especially for large scale industrialization. One is Candida antartica lipase immobilized on acrylic resin, and the other is Candida sp. 99-125 lipase immobilized on inexpensive textile membranes. However, to further reduce the cost of biodiesel production, new immobilization techniques with higher activity and stability still need to be explored. Copyright 2010 Elsevier Inc. All rights reserved.

Covalently bound conjugates of albumin and heparin: Synthesis, fractionation and characterization

NARCIS (Netherlands)

Hennink, Wim E.; Feijen, Jan; Ebert, Charles D.; Kim, Sung Wan

1983-01-01

Covalently bound conjugates of human serum albumin and heparin were prepared as compounds which could improve the blood-compatibility of polymer surfaces either by preadsorption or by covalent coupling of the conjugates onto blood contacting surfaces. The conjugates (10–16 weight % of heparin) were

Immobilization Patterns and Dynamics of Acetate-Utilizing Methanogens Immobilized in Sterile Granular Sludge in Upflow Anaerobic Sludge Blanket Reactors

Science.gov (United States)

Schmidt, Jens Ejbye; Ahring, Birgitte Kjær

1999-01-01

Sterile granular sludge was inoculated with either Methanosarcina mazeii S-6, Methanosaeta concilii GP-6, or both species in acetate-fed upflow anaerobic sludge blanket (UASB) reactors to investigate the immobilization patterns and dynamics of aceticlastic methanogens in granular sludge. After several months of reactor operation, the methanogens were immobilized, either separately or together. The fastest immobilization was observed in the reactor containing M. mazeii S-6. The highest effluent concentration of acetate was observed in the reactor with only M. mazeii S-6 immobilized, while the lowest effluent concentration of acetate was observed in the reactor where both types of methanogens were immobilized together. No changes were observed in the kinetic parameters (Ks and μmax) of immobilized M. concilii GP-6 or M. mazeii S-6 compared with suspended cultures, indicating that immobilization does not affect the growth kinetics of these methanogens. An enzyme-linked immunosorbent assay using polyclonal antibodies against either M. concilii GP-6 or M. mazeii S-6 showed significant variations in the two methanogenic populations in the different reactors. Polyclonal antibodies were further used to study the spatial distribution of the two methanogens. M. concilii GP-6 was immobilized only on existing support material without any specific pattern. M. mazeii S-6, however, showed a different immobilization pattern: large clumps were formed when the concentration of acetate was high, but where the acetate concentration was low this strain was immobilized on support material as single cells or small clumps. The data clearly show that the two aceticlastic methanogens immobilize differently in UASB systems, depending on the conditions found throughout the UASB reactor. PMID:10049862

Covalently Immobilised Cytochrome C Imaged by In Situ Scanning Tunnelling Microscopy

DEFF Research Database (Denmark)

Andersen, Jens Enevold Thaulov; Olesen, Klaus G.; Danilov, Alexey I.

1997-01-01

In situ scanning tunnelling microscopy (STM) imaging of cytochrome c (cyt c) on polycrystalline Pt surfaces and on Au(lll) was achieved first by covalent immobilisation of 3-aminopropyltriethoxysilane (3-APTS) brought to react with oxide present on the Pt surfaces. Covalently bound 3-APTS forms...

On-surface synthesis of covalent coordination polymers on micrometer scale

Institute of Scientific and Technical Information of China (English)

Mathieu Koudia; Elena Nardi; Olivier Siri; Mathieu Abel

2017-01-01

On-surface synthesis under ultrahigh vacuum provides a promising strategy to control matter at the atomic level,with important implications for the design of new two-dimensional materials having remarkable electronic,magnetic,or catalytic properties.This strategy must address the problem of limited extension of the domains due to the irreversible nature of covalent bonds,which prevents the ripening of defects.We show here that extended materials can be produced by a controlled co-deposition process.In particular,co-deposition of quinoid zwitterion molecules with iron atoms on a Ag(111) surface held at 570 K allows the formation of micrometer-sized domains based on covalent coordination bonds.This work opens up the construction of micrometer-scale single-layer covalent coordination materials under vacuum conditions.

Biodegradation of chlorobenzene using immobilized crude extracts ...

African Journals Online (AJOL)

SERVER

2007-10-04

Oct 4, 2007 ... immobilized crude extracts were reused for all other experiments and found that immobilization .... India which are of analytical reagent grade. .... 9. 60. 3. 1. Figure 3. Degradation of chlorobenzene by immobilized crude.

Use of Functionalized Carbon Nanotubes for Covalent Attachment of Nanotubes to Silicon

Science.gov (United States)

Tour, James M.; Dyke, Christopher A.; Maya, Francisco; Stewart, Michael P.; Chen, Bo; Flatt, Austen K.

2012-01-01

The purpose of the invention is to covalently attach functionalized carbon nanotubes to silicon. This step allows for the introduction of carbon nanotubes onto all manner of silicon surfaces, and thereby introduction of carbon nano - tubes covalently into silicon-based devices, onto silicon particles, and onto silicon surfaces. Single-walled carbon nanotubes (SWNTs) dispersed as individuals in surfactant were functionalized. The nano - tube was first treated with 4-t-butylbenzenediazonium tetrafluoroborate to give increased solubility to the carbon nanotube; the second group attached to the sidewall of the nanotube has a silyl-protected terminal alkyne that is de-protected in situ. This gives a soluble carbon nanotube that has functional groups appended to the sidewall that can be attached covalently to silicon. This reaction was monitored by UV/vis/NJR to assure direct covalent functionalization.

A novel, ultra sensible biosensor built by layer-by-layer covalent attachment of a receptor for diagnosis of tumor growth

International Nuclear Information System (INIS)

Uygun, Zihni Onur; Sezgintuerk, Mustafa Kemal

2011-01-01

Highlights: → Vascular Entothelial Growth Factor Receptor-1 was used as a biorecognition element as a first time in the literature. → Electrochemical impedance spectroscopy, as a measurement principle was used for analysis of VEGF-R1/VEGF interaction as a first time. → A layer-by-layer immobilization procedure enhanced the sensibility of the biosensor. → The biosensor could detect vascular endothelial growth factor in the range of 100-600 femtogram mL -1 . - Abstract: In the presented research, a novel, ultra sensitive biosensor for the impedimetric detection of vascular endothelial growth factor (VEGF) is introduced. The human vascular endothelial growth factor receptor 1 (VEGF-R1, Flt-1) was used as a biorecognition element for the first time. The immobilization of VEGF-R1 on glassy carbon electrodes was carried out using layer-by-layer covalent attachment of VEGF-R1. The electrochemical properties of the layers constructed on the electrodes were characterized by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). The differences in electron transfer resistance (R et ) between the working solution and the biosensor surface, recorded by the redox probe K 3 [Fe(CN) 6 ]/K 4 [Fe(CN) 6 ], confirmed the binding of VEGF to VEGF-R1. The new biosensor allowed a detection limit of 100 fg mL -1 with a linear range of 100-600 fg mL -1 to be obtained. The biosensor also exhibited good repeatability (with a correlation coefficient of 1.95%), and reproducibility.

Immobilized/P25/DSAT and Immobilized/Kronos/DSAT on Photocatalytic Degradation of Reactive Red 4 Under Fluorescent Light

Directory of Open Access Journals (Sweden)

Azami M. S.

2016-01-01

Full Text Available In this work, photocatalytic degradation of Reactive Red 4 (RR4 using immobilized P25 and kronos were performed under fluorescent light sources. The photocatalysis activity for both catalysts was investigated under fluorescent lamp source which consist UV and Visible light. The effect of various parameters such as initial concentration, initial pH and strenght of immobilized plate were studied. The result showed that 90% of RR4 dye was degrade in 1 hr using immobilized/kronos/DSAT at 100 mg L-1 of RR4 dye while 81% degradation was achieved by immobilized/P25/DSAT at the same condition. The lowest pH showed the higher photocatalytic activity. Hence, the effect of dye concentration and pH on the photocatalysis study can be related with the behavior of environmental pollution. The low strength showed by immobilized/P25/DSAT where it remain 37 % as compared with strength of immobilized/kronos/DSAT (52 wt.%. For the future work, the polymer binder like Polyvinyl alcohol (PVA, Polyethylene glycol (PEG, and others polymers can be apply in immobilized study to overcome the strength problem.

Continuous Packed Bed Reactor with Immobilized β-Galactosidase for Production of Galactooligosaccharides (GOS

Directory of Open Access Journals (Sweden)

Barbara Rodriguez-Colinas

2016-11-01

Full Text Available The β-galactosidase from Bacillus circulans was covalently attached to aldehyde-activated (glyoxal agarose beads and assayed for the continuous production of galactooligosaccharides (GOS in a packed-bed reactor (PBR. The immobilization was fast (1 h and the activity of the resulting biocatalyst was 97.4 U/g measured with o-nitrophenyl-β-d-galactopyranoside (ONPG. The biocatalyst showed excellent operational stability in 14 successive 20 min reaction cycles at 45 °C in a batch reactor. A continuous process for GOS synthesis was operated for 213 h at 0.2 mL/min and 45 °C using 100 g/L of lactose as a feed solution. The efficiency of the PBR slightly decreased with time; however, the maximum GOS concentration (24.2 g/L was obtained after 48 h of operation, which corresponded to 48.6% lactose conversion and thus to maximum transgalactosylation activity. HPAEC-PAD analysis showed that the two major GOS were the trisaccharide Gal-β(1→4-Gal-β(1→4-Glc and the tetrasaccharide Gal-β(1→4-Gal-β(1→4-Gal-β(1→4-Glc. The PBR was also assessed in the production of GOS from milk as a feed solution. The stability of the bioreactor was satisfactory during the first 8 h of operation; after that, a decrease in the flow rate was observed, probably due to partial clogging of the column. This work represents a step forward in the continuous production of GOS employing fixed-bed reactors with immobilized β-galactosidases.

Design-Only Conceptual Design Report: Plutonium Immobilization Plant

International Nuclear Information System (INIS)

DiSabatino, A.; Loftus, D.

1999-01-01

This design-only conceptual design report was prepared to support a funding request by the Department of Energy Office of Fissile Materials Disposition for engineering and design of the Plutonium Immobilization Plant, which will be used to immobilize up to 50 tonnes of surplus plutonium. The siting for the Plutonium Immobilization Plant will be determined pursuant to the site-specific Surplus Plutonium Disposition Environmental Impact Statement in a Plutonium Deposition Record of Decision in early 1999. This document reflects a new facility using the preferred technology (ceramic immobilization using the can-in-canister approach) and the preferred site (at Savannah River). The Plutonium Immobilization Plant accepts plutonium from pit conversion and from non-pit sources and, through a ceramic immobilization process, converts the plutonium into mineral-like forms that are subsequently encapsulated within a large canister of high-level waste glass. The final immobilized product must make the plutonium as inherently unattractive and inaccessible for use in nuclear weapons as the plutonium in spent fuel from commercial reactors and must be suitable for geologic disposal. Plutonium immobilization at the Savannah River Site uses: (1) A new building, the Plutonium Immobilization Plant, which will convert non-pit surplus plutonium to an oxide form suitable for the immobilization process, immobilize plutonium in a titanate-based ceramic form, place cans of the plutonium-ceramic forms into magazines, and load the magazines into a canister; (2) The existing Defense Waste Processing Facility for the pouring of high-level waste glass into the canisters; and (3) The Actinide Packaging and Storage Facility to receive and store feed materials. The Plutonium Immobilization Plant uses existing Savannah River Site infra-structure for analytical laboratory services, waste handling, fire protection, training, and other support utilities and services. The Plutonium Immobilization Plant

Management of immobilization and its complication for elderly.

Science.gov (United States)

Laksmi, Purwita W; Harimurti, Kuntjoro; Setiati, Siti; Soejono, Czeresna H; Aries, Wanarani; Roosheroe, Arya Govinda

2008-10-01

Increased life expectancy have an effect on the rising percentage of elderly population in Indonesia and health problem associated with the elderly, particularly immobilization. Immobilization may cause various complications, especially when it has been overlooked without any appropriate and proper medical care in keeping with the procedures. High incidence of immobilization in elderly and the life-threatening complication call for an agreement on management of immobilization and its complication. Management of immobilization needs interdisciplinary team-work cooperation, the patients and their family. The management may be commenced through a complete geriatric review, formulating functional goals and constructing therapeutic plan. Various medical conditions and external factors that may act as risk factors of immobilization as well as drugs intake that may exaggerate the immobilization should be evaluated and optimally managed. Any complication due to immobilization and other concomitant disease/condition should be recognized and managed comprehensively in order to reduce morbidity and mortality. Management of immobilization and its complications include pharmacological and non-pharmacological treatment, i.e. various mobility exercises, utilization of ambulatory device and supporting appliance for assisting patients in stand-up position, as well as the management of urinary voiding and defecation.

Excess Weapons Plutonium Immobilization in Russia

International Nuclear Information System (INIS)

Jardine, L.; Borisov, G.B.

2000-01-01

The joint goal of the Russian work is to establish a full-scale plutonium immobilization facility at a Russian industrial site by 2005. To achieve this requires that the necessary engineering and technical basis be developed in these Russian projects and the needed Russian approvals be obtained to conduct industrial-scale immobilization of plutonium-containing materials at a Russian industrial site by the 2005 date. This meeting and future work will provide the basis for joint decisions. Supporting R and D projects are being carried out at Russian Institutes that directly support the technical needs of Russian industrial sites to immobilize plutonium-containing materials. Special R and D on plutonium materials is also being carried out to support excess weapons disposition in Russia and the US, including nonproliferation studies of plutonium recovery from immobilization forms and accelerated radiation damage studies of the US-specified plutonium ceramic for immobilizing plutonium. This intriguing and extraordinary cooperation on certain aspects of the weapons plutonium problem is now progressing well and much work with plutonium has been completed in the past two years. Because much excellent and unique scientific and engineering technical work has now been completed in Russia in many aspects of plutonium immobilization, this meeting in St. Petersburg was both timely and necessary to summarize, review, and discuss these efforts among those who performed the actual work. The results of this meeting will help the US and Russia jointly define the future direction of the Russian plutonium immobilization program, and make it an even stronger and more integrated Russian program. The two objectives for the meeting were to: (1) Bring together the Russian organizations, experts, and managers performing the work into one place for four days to review and discuss their work with each other; and (2) Publish a meeting summary and a proceedings to compile reports of all the

Radiation immobilization of catalase and its application

International Nuclear Information System (INIS)

Wang Guanghui; Ha Hongfei; Wang Xia; Wu Jilan

1988-01-01

Catalase was immobilized by a chemical method on porous polyacrylamide particles produced by radiation polymerization of acrylamide monomer at low temperature (-78 0 C). Activity of immobilized catalase was enhanced distinctly by joining a chemical arm to the support. The method of recovery of catalase activity on immobilized polymer was found by soaking it in certain buffer. The treatment of H 2 O 2 both in aqueous solution and alcoholic solution by using the immobilized catalase was performed. (author)

Immobilization of carbon nanotubes on functionalized graphene film grown by chemical vapor deposition and characterization of the hybrid material

Directory of Open Access Journals (Sweden)

Prashanta Dhoj Adhikari

2014-01-01

Full Text Available We report the surface functionalization of graphene films grown by chemical vapor deposition and fabrication of a hybrid material combining multi-walled carbon nanotubes and graphene (CNT–G. Amine-terminated self-assembled monolayers were prepared on graphene by the UV-modification of oxidized groups introduced onto the film surface. Amine-termination led to effective interaction with functionalized CNTs to assemble a CNT–G hybrid through covalent bonding. Characterization clearly showed no defects of the graphene film after the immobilization reaction with CNT. In addition, the hybrid graphene material revealed a distinctive CNT–G structure and p–n type electrical properties. The introduction of functional groups on the graphene film surface and fabrication of CNT–G hybrids with the present technique could provide an efficient, novel route to device fabrication.

Thiolated polymers: evaluation of the influence of the amount of covalently attached L-cysteine to poly(acrylic acid).

Science.gov (United States)

Palmberger, Thomas F; Albrecht, Karin; Loretz, Brigitta; Bernkop-Schnürch, Andreas

2007-06-01

It was the aim of this study to investigate the influence of the amount of thiol groups being covalently attached to poly(acrylic acid) 450 kDa on its properties. Five different PAA(450)-L-cysteine conjugates (PAA(450)-Cys) were synthesized bearing 53.0 (PAA I), 113.4 (PAA II), 288.8 (PAA III), 549.1 (PAA IV) and 767.0 (PAA V) micromol immobilized thiol groups per gram polymer. Mucoadhesion studies utilizing the rotating cylinder method, tensile studies and disintegration studies were performed. Self-crosslinking properties were measured by the increase in viscosity. Permeation studies were performed on rat small intestine and Caco-2 monolayers using sodium fluorescein as model drug. Following residence times on the rotating cylinder could be identified: PAA I 3.1; PAA II 5.2; PAA III 22.0; PAA IV 33.8; PAA V 53.7; control 1.3 [h]. The disintegration time of all PAA(450)-Cys tablets was strongly dependent on the degree of thiolation of the polymer. Self-crosslinking studies showed that the different PAA(450)-Cys conjugates (3% m/v) in phosphate buffer, pH 6.8, formed intramolecular disulfide bonds. In case of Caco-2 monolayer transport studies following P(app)-values could be identified: PAA I 9.8; PAA II 10.1; PAA III 11.1; PAA IV 8.9; PAA V 8.2; control 6.4 [P(app)x10(-6), cms(-1)]. Mucoadhesive and self-crosslinking properties are strongly dependent on the degree of thiolation of the polymer and with respect to transport studies, an optimum amount of covalently attached L-cysteine could be identified.

Radioactive seed immobilization techniques for interstitial brachytherapy

International Nuclear Information System (INIS)

Yan, K.; Podder, T.; Buzurovic, I.; Hu, Y.; Dicker, A.; Valicenti, R.; Yu, Y.; Messing, E.; Rubens, D.; Sarkar, N.; Ng, W.

2008-01-01

In prostate brachytherapy, seeds can detach from their deposited sites and move locally in the pelvis or migrate to distant sites including the pulmonary and cardiac regions. Undesirable consequences of seed migration include inadequate dose coverage of the prostate and tissue irradiation effects at the site of migration. Thus, it is clinically important to develop seed immobilization techniques. We first analyze the possible causes for seed movement, and propose three potential techniques for seed immobilization: (1) surgical glue, (2) laser coagulation and (3) diathermy coagulation. The feasibility of each method is explored. Experiments were carried out using fresh bovine livers to investigate the efficacy of seed immobilization using surgical glue. Results have shown that the surgical glue can effectively immobilize the seeds. Evaluation of the radiation dose distribution revealed that the non-immobilized seed movement would change the planned isodose distribution considerably; while by using surgical glue method to immobilize the seeds, the changes were negligible. Prostate brachytherapy seed immobilization is necessary and three alternative mechanisms are promising for addressing this issue. Experiments for exploring the efficacy of the other two proposed methods are ongoing. Devices compatible with the brachytherapy procedure will be designed in future. (orig.)

Characteristics of Immobilized Urease on Grafted Alginate Bead Systems

Directory of Open Access Journals (Sweden)

Enas N. Danial

2015-04-01

Full Text Available This study evaluated the biological importance of immobilized urease enzyme over the free urease. The support material used for urease immobilization was alginate. Generally, the immobilization of urease in alginate gel showed a marked increase in Km and Vmax. However, the immobilized urease showed higher thermal stability than that of free enzyme. The rate of thermal inactivation of the immobilized enzyme decreased due to entrapment in gel matrix. Also, the activity of the immobilized urease was more stable in retention than that of the free enzyme during the storage in solution, although the activity of the immobilized enzyme was lower in comparison with the free enzyme. A stable immobilized system and long storage life are convenient for applications that would not be feasible with a soluble enzyme system. These results highlighted the technical and biochemical benefits of immobilized urease over the free enzyme.

Activities of lectins and their immobilized derivatives in detergent solutions. Implications on the use of lectin affinity chromatography for the purification of membrane glycoproteins.

Science.gov (United States)

Lotan, R; Beattie, G; Hubbell, W; Nicolson, G L

1977-05-03

The effects of several commonly used detergents on the saccharide-binding activities of lectins were investigated using lectin-mediated agglutination of formalin-fixed erythrocytes and affinity chromatography of glycoproteins on columns of lectins immobilized on polyacrylic hydrazide-Sepharose. In the hemagglutination assays, Ricinus communis I (RCA1) and II (RCAII), concanavalin A (Con A), and the agglutinins from peanut (PNA), soybean (SBA), wheat germ (WGA), and Limulus polyphemus (LPA) were tested with several concentrations of switterionic, cationic, anionic, and nonionic detergents. It was found that increasing detergent concentrations eventually affected hemagglutination titers in both test and control samples, and the highest detergent concentrations not affecting lectin hemagglutinating activities were determined. The effects of detergents on specific binding of [3H]fetuin and asialo[3H]fetuin to and elution from columns of immobilized lectins were less severe when compared with lectins in solution, suggesting that the lectins are stabilized by covalent attachment to agarose beads. Nonionic detergents did not affect the binding efficiency of the immobilized lectins tested at concentrations used for membrane solubilization while cationic and zwitterionic detergents caused significant inhibition of Con A- and SBA-Sepharose activities. In sodium deoxycholate (greater than 1%) only RCAI-Sepharose retained its activity, whereas the activities of the other lectins were reduced dramatically. Low concentrations of sodium dodecyl sulfate (0.05%) inhibited only the activity of immobilized SBA, but at higher concentration (0.1%) and prolonged periods of incubation (16 h, 23 degrees C) most of the lectins were inactivated. These data are compared with previous reports on the use of detergents in lectin affinity chromatography, and the conditions for the optimal use of detergents are detailed.

ORF Alignment: NC_004307 [GENIUS II[Archive

Lifescience Database Archive (English)

Full Text Available nosidase (sucrase/invertase); possible inulinase ... [Bifidobacterium longum NCC2705] gb|AAN23970.1| ... ... beta-fructofuranosidase (sucrase/invertase); possible ... inulinase [Bifidobacterium longu

Growth, ethanol production, and inulinase activity on various inulin substrates by mutant Kluyveromyces marxianus strains NRRL Y-50798 and NRRL Y-50799.

Science.gov (United States)

Galindo-Leva, Luz Ángela; Hughes, Stephen R; López-Núñez, Juan Carlos; Jarodsky, Joshua M; Erickson, Adam; Lindquist, Mitchell R; Cox, Elby J; Bischoff, Kenneth M; Hoecker, Eric C; Liu, Siqing; Qureshi, Nasib; Jones, Marjorie A

2016-07-01

Economically important plants contain large amounts of inulin. Disposal of waste resulting from their processing presents environmental issues. Finding microorganisms capable of converting inulin waste to biofuel and valuable co-products at the processing site would have significant economic and environmental impact. We evaluated the ability of two mutant strains of Kluyveromyces marxianus (Km7 and Km8) to utilize inulin for ethanol production. In glucose medium, both strains consumed all glucose and produced 0.40 g ethanol/g glucose at 24 h. In inulin medium, Km7 exhibited maximum colony forming units (CFU)/mL and produced 0.35 g ethanol/g inulin at 24 h, while Km8 showed maximum CFU/mL and produced 0.02 g ethanol/g inulin at 96 h. At 24 h in inulin + glucose medium, Km7 produced 0.40 g ethanol/g (inulin + glucose) and Km8 produced 0.20 g ethanol/g (inulin + glucose) with maximum CFU/mL for Km8 at 72 h, 40 % of that for Km7 at 36 h. Extracellular inulinase activity at 6 h for both Km7 and Km8 was 3.7 International Units (IU)/mL.

Matrigel immobilization on the shish-kebab structured poly(ɛ-caprolactone) nanofibers for skin tissue engineering

Science.gov (United States)

Jing, Xin; Mi, Hao-Yang; Peng, Xiang-Fang; Turng, Lih-Sheng

2016-03-01

Surface properties of tissue engineering scaffolds such as topography, hydrophilicity, and functional groups play a vital role in cell adhesion, migration, proliferation, and apoptosis. First, poly(ɛ-caprolactone) (PCL) shish-kebab scaffolds (PCL-SK), which feature a three-dimensional structure comprised of electrospun PCL nanofibers covered by periodic, self-induced PCL crystal lamellae on the surface, was created to mimic the nanotopography of native collagen fibrils in the extracellular matrix (ECM). Second, matrigel was covalently immobilized on the surface of alkaline hydrolyzed PCL-SK scaffolds to enhance their hydrophilicity. This combined approach not only mimics the nanotopography of native collagen fibrils, but also simulates the surface features of collagen fibrils for cell growth. To investigate the viability of such scaffolds, HEF1 fibroblast cell assays were conducted and the results revealed that the nanotopography of the PCL-SK scaffolds facilitated cell adhesion and proliferation. The matrigel functionalization on PCL-SK scaffolds further enhanced cellular response, which suggested elevated biocompatibility and greater potential for skin tissue engineering applications.

Matrigel immobilization on the shish-kebab structured poly(ε-caprolactone) nanofibers for skin tissue engineering

Energy Technology Data Exchange (ETDEWEB)

Jing, Xin, E-mail: jingxinscut@gmail.com; Mi, Hao-Yang [National Engineer Research Center of Novel Equipment for Polymer Processing, The Key Laboratory of Polymer Processing Engineering of Ministry of Education, South China University of Technology, Guangzhou, 510640 (China); Wisconsin Institutes for Discovery, University of Wisconsin-Madison, 53715 (United States); Peng, Xiang-Fang, E-mail: pmxfpeng@scut.edu.cn, E-mail: turng@engr.wisc.edu [National Engineer Research Center of Novel Equipment for Polymer Processing, The Key Laboratory of Polymer Processing Engineering of Ministry of Education, South China University of Technology, Guangzhou, 510640 (China); Turng, Lih-Sheng, E-mail: pmxfpeng@scut.edu.cn, E-mail: turng@engr.wisc.edu [Wisconsin Institutes for Discovery, University of Wisconsin-Madison, 53715 (United States)

2016-03-09

Surface properties of tissue engineering scaffolds such as topography, hydrophilicity, and functional groups play a vital role in cell adhesion, migration, proliferation, and apoptosis. First, poly(ε-caprolactone) (PCL) shish-kebab scaffolds (PCL-SK), which feature a three-dimensional structure comprised of electrospun PCL nanofibers covered by periodic, self-induced PCL crystal lamellae on the surface, was created to mimic the nanotopography of native collagen fibrils in the extracellular matrix (ECM). Second, matrigel was covalently immobilized on the surface of alkaline hydrolyzed PCL-SK scaffolds to enhance their hydrophilicity. This combined approach not only mimics the nanotopography of native collagen fibrils, but also simulates the surface features of collagen fibrils for cell growth. To investigate the viability of such scaffolds, HEF1 fibroblast cell assays were conducted and the results revealed that the nanotopography of the PCL-SK scaffolds facilitated cell adhesion and proliferation. The matrigel functionalization on PCL-SK scaffolds further enhanced cellular response, which suggested elevated biocompatibility and greater potential for skin tissue engineering applications.

Immobilization of Mortierella vinacea cells by radiation polymerization

International Nuclear Information System (INIS)

Kumakura, M.; Kaetsu, I.

1983-01-01

Immobilization of Mortierella vinacea cells, which contain active α-galactosidase, by radiation polymerization at low temperatures was studied. The durability of the enzymatic activity of the immobilized cells obtained with hydrophilic monomers was affected by the concentrations of the cells and monomer in which optimum conditions were observed. The enzymatic activity of the immobilized cells obtained with hydrophilic monomers was compared to that of hydrophobic monomers. Michaelis constants of the immobilized cells varied with monomer concentration. The effect of addition of porous solid substances on the immobilization of the cells was studied

The covalent effect on the energy levels of d3 ions in tetragonal compounds

International Nuclear Information System (INIS)

Li, Dong-Yang; Du, Mao-Lu

2015-01-01

For d 3 ions in covalent compounds with tetragonal symmetry, this paper presents a complete energy matrix, in which the different covalence of t 2 and e orbitals is considered not only in the electrostatic repulsions part of energy matrix elements but also in the crystal-field potential part of energy matrix elements. With taking and no taking the crystal field parameter B 00 0 into account, the effect of covalence on the energy levels of d 3 ions system were investigated, respectively. The investigation shows that it is very necessary for considering the different covalence of t 2 and e orbitals in both electrostatic repulsions part and crystal-field potential part when the optical properties of d 3 ions in strong covalent compounds with tetragonal symmetry is investigated. On the other hand, the crystal field parameter B 00 0 has a significant effect on the energy levels, and should be considered in investigations of d 3 ions in strong covalent compounds with tetragonal symmetry. Application to calculating the energy levels for Co 2+ in CdGa 2 Se 4 , the calculated results are in agreement with the experiment data

Macrocyclic receptors immobilized to monodisperse porous polymer particles by chemical grafting and physical impregnation for strontium capture: a comparative study.

Science.gov (United States)

Song, Yang; Du, Yi; Lv, Dachao; Ye, Gang; Wang, Jianchen

2014-06-15

Separation of strontium is of great significance for radioactive waste treatment and environmental remediation after nuclear accidents. In this work, a novel class of adsorbent (Crown-g-MPPPs) was synthesized by chemical grafting a macrocyclic ether receptor to monodisperse porous polymer particles (MPPPs) for strontium adsorption. Meanwhile, a counterpart material (Crown@MPPPs) with the receptor molecules immobilized to the MPPPs substrate by physical impregnation was prepared. To investigate how the immobilization manner and distribution of the receptors influence the adsorption ability, a comparative study on the adsorption behaviour of the two materials towards Sr(II) in HNO3 media was accomplished. Due to the shorter diffusion path and covalently-bonded structure, Crown-g-MPPPs showed faster adsorption kinetics and better stability for cycle use. While Crown@MPPPs had the advantages of facile synthesis and higher adsorption capacity, owing to the absence of conformational constraint to form complexation with Sr(II). Kinetic functions (Lagergren pseudo-first-order/pseudo-second-order functions) and adsorption isotherm models (Langmuir/Freundlich models) were used to fit the experimental data and examine the adsorption mechanism. On this basis, a chromatographic process was proposed by using Crown@MPPPs for an effective separation of Sr(II) (91%) in simulated high level liquid waste (HLLW). Copyright © 2014 Elsevier B.V. All rights reserved.

Treatment and immobilization of intermediate level radioactive wastes

International Nuclear Information System (INIS)

Lerch, R.E.; Greenhalgh, W.O.; Partridge, J.A.; Richardson, G.L.

1977-01-01

This paper discusses a new program underway to develop and demonstrate treatment and immobilization technologies for intermediate level wastes (ILW) generated in the nuclear fuel cycle. Initial work has defined the sources, quantities and types of wastes which comprise ILW. Laboratory studies are underway to define treatment technologies for liquid ILW which contains volatile contaminants and to define immobilization parameters for the residues resulting from treatment of ILW. Immobilization agents initially being evaluated for the various residues include cement, urea-formaldehyde, and bitumen although other immobilization agents will be studied. The program also includes development of acceptable test procedures for the final immobilized products as well as development of proposed criteria for storage, transportation, and disposal of the immobilized ILW. 20 figures, 10 tables

Preparation and immobilization of noble metal nanoparticles for plasmonic solar cells

Energy Technology Data Exchange (ETDEWEB)

Wang, Ruoli; Pitzer, Martin; Hu, DongZhi; Schaadt, Daniel M. [Institut fuer Angewandte Physik, Karlsruher Institut fuer Technologie (KIT), Karlsruhe (Germany); DFG Centrum fuer Funktionelle Nanostrukturen (CFN), KIT (Germany); Fruk, Ljiljana [DFG Centrum fuer Funktionelle Nanostrukturen (CFN), KIT (Germany)

2011-07-01

Thin-film solar cells are of high interest due to good electrical properties and low material consumption. Traditional thin-film cells, however, have considerable transmission losses because of the reduced absorption volume. A promising way to enhance absorption in the active layer is the light-trapping by plasmonic nanostructures. Metallic nanoparticles have in particular shown large enhancement of the photocurrent in thin-film devices. In this poster, we present preparation of Au,Ag and Pt nanoparticles by polyol method and seed mediated methods for use in plasmonic solar cells. Polyol method typically uses ethylene glycol as the solvent and reducing agent,and in seed-mediated synthesis small nanoparticle seeds are first prepared and then used to promote the growth of different shapes of nanoparticles. We particularly focus on the use of nanocubes and nanospheres for solar cell design. Following the nanoparticle preparation, a new method to immobilize particles on GaAs surfaces via covalent chemical bonds has been developed which prevents agglomerations and allows control of the surface density. Photocurrent spectra of GaAs pin solar cells with and without particles have been recorded. These measurements show the dependence of the photocurrent enhancement on particle material, shape and density.

Covalent biofunctionalization of silicon nitride surfaces

NARCIS (Netherlands)

Arafat, A.; Giesbers, M.; Rosso, M.; Sudhölter, E.J.R.; Schroën, C.G.P.H.; White, R.G.; Li Yang,; Linford, M.R.; Zuilhof, H.

2007-01-01

Covalently attached organic monolayers on etched silicon nitride (SixN4; x 3) surfaces were prepared by reaction of SixN4-coated wafers with neat or solutions of 1-alkenes and 1-alkynes in refluxing mesitylene. The surface modification was monitored by measurement of the static water contact angle,

Evidence of significant covalent bonding in Au(CN)(2)(-).

Science.gov (United States)

Wang, Xue-Bin; Wang, Yi-Lei; Yang, Jie; Xing, Xiao-Peng; Li, Jun; Wang, Lai-Sheng

2009-11-18

The Au(CN)(2)(-) ion is the most stable Au compound known for centuries, yet a detailed understanding of its chemical bonding is still lacking. Here we report direct experimental evidence of significant covalent bonding character in the Au-C bonds in Au(CN)(2)(-) using photoelectron spectroscopy and comparisons with its lighter congeners, Ag(CN)(2)(-) and Cu(CN)(2)(-). Vibrational progressions in the Au-C stretching mode were observed for all detachment transitions for Au(CN)(2)(-), in contrast to the atomic-like transitions for Cu(CN)(2)(-), revealing the Au-C covalent bonding character. In addition, rich electronic structural information was obtained for Au(CN)(2)(-) by employing 118 nm detachment photons. Density functional theory and high-level ab initio calculations were carried out to understand the photoelectron spectra and obtain insight into the nature of the chemical bonding in the M(CN)(2)(-) complexes. Significant covalent character in the Au-C bonding due to the strong relativistic effects was revealed in Au(CN)(2)(-), consistent with its high stability.

Adsorption of Cu (II), Pb (II) and Cr (VI) from aqueous solutions using black wattle tannin-immobilized nanocellulose.

Science.gov (United States)

Xu, Qinghua; Wang, Yulu; Jin, Liqiang; Wang, Yu; Qin, Menghua

2017-10-05

A novel nanocomposite based on black wattle (BW) tannin and nanocellulose was prepared and applied in heavy metal ions adsorptive removal from aqueous solutions. Firstly, nanocrystalline cellulose was oxidized by sodium periodate to get dialdehyde nanocellulose (DANC). BW tannin was then covalently immobilized onto DANC, which was used as both the matrix and crosslinker, to obtain tannin-nanocellulose (TNCC) composite. The resulting nanocomposite was characterized using FTIR, AFM, and TG. The successful immobilization was confirmed by the chromogenic reaction between FeCl 3 and TNCC and FT-IR analysis. AFM images revealed that TNCC was ellipsoidal particles with lengths ranging from 100-400nm. Zeta potential measurement showed that TNCC was negative charged at a pH range from 1-12. Compared to the original tannin, the thermal stability of TNCC was slightly increased by the addition of nanocellulose. TNCC demonstrated the maximum adsorption efficiency at pH2 for Cr(VI) and pH 6 for Cu(II) and Pb(II), respectively. The adsorption for these three metal ions followed pseudo second-order kinetics, indicating the chemisorption nature. The adsorption isotherms all fitted well with the Sips model, and the calculated maximum adsorption capacities were 51.846mgg -1 , 53.371mgg -1 and 104.592mgg -1 for Cu(II), Pb(II) and Cr (VI), respectively. Copyright © 2017 Elsevier B.V. All rights reserved.

Covalent-ionically cross-linked polyetheretherketone proton exchange membrane for direct methanol fuel cell

CSIR Research Space (South Africa)

Luo, H

2010-08-01

Full Text Available cross-linked PEEK-WC membrane, this covalent-ionically cross-linked PEEK-WC membrane exhibits extremely reduced water uptake and methanol permeability, but just slightly sacrificed proton conductivity. The proton conductivity of the covalent...

共价键的离子化%Ionization of Covalent Bond

Institute of Scientific and Technical Information of China (English)

王稼国; 荆西平

2017-01-01

本文用化学键理论分析和推导了共价键离子化的几种方式,包括金属和非金属单质中共价键的诱导离子化、化合物中共价键的降温和自诱导离子化以及含氢化合物和金属化合物的诱导离子化.从能量角度分析了离子化趋势的规律性,并且讨论了共价键的离子化的一些重要应用.%Several ionization patterns of covalent bond,including induced-ionization of covalent bond in metals and nonmetals,induced-ionization of covalent bond in hydrogen compounds and metal compounds,lowering temperature and self-induced ionization of compound,and so on,were dedueed and analyzed by using chemical bond theory.The trend of ionization was also analyzed on energy changing and several important applications of the ionization of covalent bond were discussed.

Properties of immobilized papain by radiation polymerization

International Nuclear Information System (INIS)

Kumakura, Minoru; Kaetsu, Isao

1984-01-01

Papain was immobilized by the radiation polymerization of various monomers at low temperatures and the effects of the polymer matrix on the enzyme activity and thermal stability of the immobilized enzymes were studied. The activity of the immobilized enzymes prepared from monofunctional (acrylate and methacrylate) monomers was higher than that from bifunctional (bismethacrylate) monomers and that from polyoxyethylene dimethacrylate monomers increased with an increase in the number of oxyethylene units. The thermal stability of the immobilized enzymes prepared from hydrophilic monomers was higher than that from hydrophobic monomers and increased markedly with increasing monomer concentration. (author)

Predominantly ligand guided non-covalently linked assemblies of ...

Indian Academy of Sciences (India)

JUBARAJ B BARUAH

2018-05-12

May 12, 2018 ... Abstract. Various non-covalently linked inorganic self-assemblies formed by the supramolecular interacting .... metal-organic frameworks.59 Inorganic chemists rou- ...... two-dimensional organic–inorganic layered perovskite.

A glucose biosensor based on direct electron transfer of glucose oxidase immobilized onto glassy carbon electrode modified with nitrophenyl diazonium salt

International Nuclear Information System (INIS)

Nasri, Zahra; Shams, Esmaeil

2013-01-01

Graphical abstract: - Abstract: This study reports a novel, simple and fast approach for construction of a highly stable glucose biosensor based on the immobilization of glucose oxidase (GOx) onto a glassy carbon electrode (GCE) electrografted with 4-aminophenyl (AP) by diazonium chemistry. Aminophenyl was used as cross-linker for covalent attachment of glucose oxidase to the electrode surface. Cyclic voltammograms of the GOx-modified GCE in phosphate buffer solution exhibited a pair of well-defined redox peaks, attesting the direct electron transfer (DET) of GOx with the underlying electrode. The proposed biosensor could be used to detect glucose based on the consumption of O 2 with the oxidation of glucose catalyzed by GOx and exhibited a wide linear range of glucose from 0.05 mM to 4.5 mM and low detection limit of 10 μM. The surface coverage of active GOx, heterogeneous electron transfer rate constant (k s ) and Michaelis–Menten constant (K M ) of immobilized GOx were 1.23 × 10 −12 mol cm −2 , 4.25 s −1 and 2.95 mM, respectively. The great stability of this biosensor, technically simple and possibility of preparation at short period of time make this method suitable for fabrication of low-cost glucose biosensors

Immobilization of Glucose Oxidase on a Carbon Nanotubes/Dendrimer-Ferrocene Modified Electrode for Reagentless Glucose Biosensing.

Science.gov (United States)

Zhou, Juan; Li, Huan; Yang, Huasong; Cheng, Hui; Lai, Guosong

2017-01-01

Ferrocene-grafted dendrimer was covalently linked to the surface of a carbon nanotubes (CNTs)-chitosan (CS) nanocomposite modified electrode for immobilizing high-content glucose oxidase (GOx), which resulted in the successful development a novel reagentless glucose biosensor. Electrochemical impedance spectroscopy, cyclic voltammetry, and amperometry were used to characterize the preparation process and the enzymatically catalytic response of this biosensor. Due to the excellent electron transfer acceleration of the CNTs and the high-content loading of the GOx biomolecule and ferrocene mediator on the electrode matrix, this biosensor showed excellent analytical performance such as fast response time less than 10 s, wide linear range from 0.02 to 2.91 mM and low detection limit down to 7.5 μM as well as satisfactory stability and reproducibility toward the amperometric glucose determination. In addition, satisfactory result was obtained when it was used for the glucose measurements in human blood samples. Thus this biosensor provides great potentials for practical applications.

Covalently Connecting Crystal Grains with Polyvinylammonium Carbochain Backbone To Suppress Grain Boundaries for Long-Term Stable Perovskite Solar Cells.

Science.gov (United States)

Li, Han; Liang, Chao; Liu, Yingliang; Zhang, Yiqiang; Tong, Jincheng; Zuo, Weiwei; Xu, Shengang; Shao, Guosheng; Cao, Shaokui

2017-02-22

Grain boundaries act as rapid pathways for nonradiative carrier recombination, anion migration, and water corrosion, leading to low efficiency and poor stability of organometal halide perovskite solar cells (PSCs). In this work, the strategy suppressing the crystal grain boundaries is applied to improve the photovoltaic performance, especially moisture-resistant stability, with polyvinylammonium carbochain backbone covalently connecting the perovskite crystal grains. This cationic polyelectrolyte additive serves as nucleation sites and template for crystal growth of MAPbI 3 and afterward the immobilized adjacent crystal grains grow into the continuous compact, pinhole-free perovskite layer. As a result, the unsealed PSC devices, which are fabricated under low-temperature fabrication protocol with a proper content of polymer additive PVAm·HI, currently exhibit the maximum efficiency of 16.3%. Remarkably, these unsealed devices follow an "outside-in" corrosion mechanism and respectively retain 92% and 80% of the initial PCE value after being exposed under ambient environment for 50 days and 100 days, indicating the superiority of carbochain polymer additives in solving the long-term stability problem of PSCs.

Molecular electrostatic potential analysis of non-covalent complexes

Indian Academy of Sciences (India)

Chemical Sciences and Technology Division and Academy of Scientific & Innovative Research (AcSIR), ... workers proposed the electrostatic-covalent model of hydrogen bonding. ..... tain degree of electron donation and acceptance occurs.

Novel immobilization process of a thermophilic catalase: efficient purification by heat treatment and subsequent immobilization at high temperature.

Science.gov (United States)

Xu, Juan; Luo, Hui; López, Claudia; Xiao, Jing; Chang, Yanhong

2015-10-01

The main goal of the present work is to investigate a novel process of purification and immobilization of a thermophilic catalase at high temperatures. The catalase, originated from Bacillus sp., was overexpressed in a recombinant Escherichia coli BL21(DE3)/pET28-CATHis and efficiently purified by heat treatment, achieving a threefold purification. The purified catalase was then immobilized onto an epoxy support at different temperatures (25, 40, and 55 °C). The immobilizate obtained at higher temperatures reached its maximum activity in a shorter time than that obtained at lower temperatures. Furthermore, immobilization at higher temperatures required a lower ionic strength than immobilization at lower temperatures. The characteristics of immobilized enzymes prepared at different temperatures were investigated. The high-temperature immobilizate (55 °C) showed the highest thermal stability, followed by the 40 °C immobilizate. And the high-temperature immobilizate (55 °C) had slightly higher operational stability than the 25 °C immobilizate. All of the immobilized catalase preparations showed higher stability than the free enzyme at alkaline pH 10.0, while the alkali resistance of the 25 °C immobilizate was slightly better than that of the 40 and 55 °C immobilizates.

Glucose Oxidase Immobilization on TMAH-Modified Bentonite

Directory of Open Access Journals (Sweden)

Ruth Chrisnasari

2015-03-01

Full Text Available The influence of bentonite modification by tetramethyl ammonium hydroxide (TMAH on its capability to immobilize glucose oxidase (GOX was studied. Modification of bentonite was conducted by the adding of 0-5% (v/v TMAH. The observed results show that the different concentrations of TMAH affect the percentage of immobilized enzyme. The results of this study show that the best concentration of TMAH is 5% (v/v which can immobilize up to 84.71% of GOX. X-ray diffraction (XRD and Fourier Transforms Infrared Spectroscopy (FTIR studies have been carried out to observe the structural changes in bentonite due to TMAH modification. The obtained immobilized GOX show the optimum catalytic activity on reaction temperature of 40-50 °C and pH of 7. The immobilized GOX kinetics at the optimum conditions determined the Km and Vmax value to be 4.96x10-2 mM and 4.99x10-3 mM.min-1 respectively. In addition, the immobilized GOX on TMAH-modified bentonite is stable enough so it could be re-used six times before its activity decreased by 39.44%.

Biotechnological production of vanillin using immobilized enzymes.

Science.gov (United States)

Furuya, Toshiki; Kuroiwa, Mari; Kino, Kuniki

2017-02-10

Vanillin is an important and popular plant flavor, but the amount of this compound available from plant sources is very limited. Biotechnological methods have high potential for vanillin production as an alternative to extraction from plant sources. Here, we report a new approach using immobilized enzymes for the production of vanillin. The recently discovered oxygenase Cso2 has coenzyme-independent catalytic activity for the conversion of isoeugenol and 4-vinylguaiacol to vanillin. Immobilization of Cso2 on Sepabeads EC-EA anion-exchange carrier conferred enhanced operational stability enabling repetitive use. This immobilized Cso2 catalyst allowed 6.8mg yield of vanillin from isoeugenol through ten reaction cycles at a 1mL scale. The coenzyme-independent decarboxylase Fdc, which has catalytic activity for the conversion of ferulic acid to 4-vinylguaiacol, was also immobilized on Sepabeads EC-EA. We demonstrated that the immobilized Fdc and Cso2 enabled the cascade synthesis of vanillin from ferulic acid via 4-vinylguaiacol with repetitive use of the catalysts. This study is the first example of biotechnological production of vanillin using immobilized enzymes, a process that provides new possibilities for vanillin production. Copyright © 2016 Elsevier B.V. All rights reserved.

Binding matter with antimatter: the covalent positron bond.

Science.gov (United States)

Charry, Jorge Alfonso; Varella, Marcio T Do N; Reyes, Andrés

2018-05-16

We report sufficient theoretical evidence of the energy stability of the e⁺H₂²⁻ molecule, formed by two H⁻ anions and one positron. Analysis of the electronic and positronic densities of the latter compound undoubtedly points out the formation of a positronic covalent bond between the otherwise repelling hydride anions. The lower limit for the bonding energy of the e⁺H₂²⁻ molecule is 74 kJ/mol (0.77 eV), accounting for the zero-point vibrational correction. The formation of a non electronic covalent bond is fundamentally distinct from positron attachment to stable molecules, as the latter process is characterized by a positron affinity, analogous to the electron affinity. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Antimicrobial activity of immobilized lactoferrin and lactoferricin.

Science.gov (United States)

Chen, Renxun; Cole, Nerida; Dutta, Debarun; Kumar, Naresh; Willcox, Mark D P

2017-11-01

Lactoferrin and lactoferricin were immobilized on glass surfaces via two linkers, 4-azidobenzoic acid (ABA) or 4-fluoro-3-nitrophenyl azide (FNA). The resulting surfaces were characterized by X-ray photoelectron spectroscopy (XPS) and contact angle measurements. The antimicrobial activity of the surfaces was determined using Pseudomonas aeruginosa and Staphylococcus aureus strains by fluorescence microscopy. Lactoferrin and lactoferricin immobilization was confirmed by XPS showing significant increases (p lactoferricin immobilized on glass significantly (p lactoferricin were successfully immobilized on glass surfaces and showed promising antimicrobial activity against pathogenic bacteria. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 2612-2617, 2017. © 2016 Wiley Periodicals, Inc.

Covalent modification of graphene and graphite using diazonium chemistry: tunable grafting and nanomanipulation.

Science.gov (United States)

Greenwood, John; Phan, Thanh Hai; Fujita, Yasuhiko; Li, Zhi; Ivasenko, Oleksandr; Vanderlinden, Willem; Van Gorp, Hans; Frederickx, Wout; Lu, Gang; Tahara, Kazukuni; Tobe, Yoshito; Uji-I, Hiroshi; Mertens, Stijn F L; De Feyter, Steven

2015-05-26

We shine light on the covalent modification of graphite and graphene substrates using diazonium chemistry under ambient conditions. We report on the nature of the chemical modification of these graphitic substrates, the relation between molecular structure and film morphology, and the impact of the covalent modification on the properties of the substrates, as revealed by local microscopy and spectroscopy techniques and electrochemistry. By careful selection of the reagents and optimizing reaction conditions, a high density of covalently grafted molecules is obtained, a result that is demonstrated in an unprecedented way by scanning tunneling microscopy (STM) under ambient conditions. With nanomanipulation, i.e., nanoshaving using STM, surface structuring and functionalization at the nanoscale is achieved. This manipulation leads to the removal of the covalently anchored molecules, regenerating pristine sp(2) hybridized graphene or graphite patches, as proven by space-resolved Raman microscopy and molecular self-assembly studies.

Building high-coverage monolayers of covalently bound magnetic nanoparticles

Energy Technology Data Exchange (ETDEWEB)

Williams, Mackenzie G.; Teplyakov, Andrew V., E-mail: andrewt@udel.edu

2016-12-01

Graphical abstract: - Highlights: • A method for forming a layer of covalently bound nanoparticles is offered. • A nearly perfect monolayer of covalently bound magnetic nanoparticles was formed on gold. • Spectroscopic techniques confirmed covalent binding by the “click” reaction. • The influence of the functionalization scheme on surface coverage was investigated. - Abstract: This work presents an approach for producing a high-coverage single monolayer of magnetic nanoparticles using “click chemistry” between complementarily functionalized nanoparticles and a flat substrate. This method highlights essential aspects of the functionalization scheme for substrate surface and nanoparticles to produce exceptionally high surface coverage without sacrificing selectivity or control over the layer produced. The deposition of one single layer of magnetic particles without agglomeration, over a large area, with a nearly 100% coverage is confirmed by electron microscopy. Spectroscopic techniques, supplemented by computational predictions, are used to interrogate the chemistry of the attachment and to confirm covalent binding, rather than attachment through self-assembly or weak van der Waals bonding. Density functional theory calculations for the surface intermediate of this copper-catalyzed process provide mechanistic insight into the effects of the functionalization scheme on surface coverage. Based on this analysis, it appears that steric limitations of the intermediate structure affect nanoparticle coverage on a flat solid substrate; however, this can be overcome by designing a functionalization scheme in such a way that the copper-based intermediate is formed on the spherical nanoparticles instead. This observation can be carried over to other approaches for creating highly controlled single- or multilayered nanostructures of a wide range of materials to result in high coverage and possibly, conformal filling.

Immobilization thresholds of electrofishing relative to fish size

Science.gov (United States)

Dolan, C.R.; Miranda, L.E.

2003-01-01

Fish size and electrical waveforms have frequently been associated with variation in electrofishing effectiveness. Under controlled laboratory conditions, we measured the electrical power required by five electrical waveforms to immobilize eight fish species of diverse sizes and shapes. Fish size was indexed by total body length, surface area, volume, and weight; shape was indexed by the ratio of body length to body depth. Our objectives were to identify immobilization thresholds, elucidate the descriptors of fish size that were best associated with those immobilization thresholds, and determine whether the vulnerability of a species relative to other species remained constant across electrical treatments. The results confirmed that fish size is a key variable controlling the immobilization threshold and further suggested that the size descriptor best related to immobilization is fish volume. The peak power needed to immobilize fish decreased rapidly with increasing fish volume in small fish but decreased slowly for fish larger than 75-100 cm 3. Furthermore, when we controlled for size and shape, different waveforms did not favor particular species, possibly because of the overwhelming effect of body size. Many of the immobilization inconsistencies previously attributed to species might simply represent the effect of disparities in body size.

Covalently bound molecular states in beryllium and carbon isotopes

International Nuclear Information System (INIS)

Wolfram von, Oertzen; Hans-Gerhard, Bohlen; Wolfram von, Oertzen

2003-01-01

Nuclear clustering in N=Z nuclei has been studied since many decades. States close to the decay thresholds, as described by the Ikeda diagram, are of particular interest. Recent studies in loosely bound systems, as observed with neutron-rich nuclei has revived the interest in cluster structures in nuclei, with additional valence neutrons, which give rise to pronounced covalent molecular structures. The Beryllium isotopes represent the first example of such unique states in nuclear physics with extreme deformations. In the deformed shell model these are referred to as super- and hyper-deformation. These states can be described explicitly by molecular concepts, with neutrons in covalent binding orbits. Examples of recent experiments performed at the HMI-Berlin demonstrating the molecular structure of the rotational bands in Beryllium isotopes are presented. Further work on chain states (nuclear polymers) in the carbon isotopes is in progress, these are the first examples of deformed structures in nuclei with an axis ratio of 3:1. A threshold diagram with clusters bound via neutrons in covalent molecular configurations can be established, which can serve as a guideline for future work. (authors)

Immobilization of yeast cells by radiation-induced polymerization

International Nuclear Information System (INIS)

Fujimura, T.; Kaetsu, I.

1982-01-01

Radiation-induced polymerization method was applied to the immobilization of yeast cells. The effects of irradiation, cooling and monomer, which are neccessary for polymerization, were recovered completely by subsequent aerobical incubation of yeast cells. The ethanol productive in immobilized yeast cells increased with the increase of aerobical incubation period. The growth of yeast cells in immobilized yeast cells was indicated. The maximum ethanol productivity in immobilized yeast cell system was around three times as much as that in free yeast cell system. (orig.)

New developments for the site-specific attachment of protein to surfaces

Energy Technology Data Exchange (ETDEWEB)

Camarero, J A

2005-05-12

Protein immobilization on surfaces is of great importance in numerous applications in biology and biophysics. The key for the success of all these applications relies on the immobilization technique employed to attach the protein to the corresponding surface. Protein immobilization can be based on covalent or noncovalent interaction of the molecule with the surface. Noncovalent interactions include hydrophobic interactions, hydrogen bonding, van der Waals forces, electrostatic forces, or physical adsorption. However, since these interactions are weak, the molecules can get denatured or dislodged, thus causing loss of signal. They also result in random attachment of the protein to the surface. Site-specific covalent attachment of proteins onto surfaces, on the other hand, leads to molecules being arranged in a definite, orderly fashion and uses spacers and linkers to help minimize steric hindrances between the protein surface. This work reviews in detail some of the methods most commonly used as well as the latest developments for the site-specific covalent attachment of protein to solid surfaces.

Ceramic membrane microfilter as an immobilized enzyme reactor.

Science.gov (United States)

Harrington, T J; Gainer, J L; Kirwan, D J

1992-10-01

This study investigated the use of a ceramic microfilter as an immobilized enzyme reactor. In this type of reactor, the substrate solution permeates the ceramic membrane and reacts with an enzyme that has been immobilized within its porous interior. The objective of this study was to examine the effect of permeation rate on the observed kinetic parameters for the immobilized enzyme in order to assess possible mass transfer influences or shear effects. Kinetic parameters were found to be independent of flow rate for immobilized penicillinase and lactate dehydrogenase. Therefore, neither mass transfer nor shear effects were observed for enzymes immobilized within the ceramic membrane. Both the residence time and the conversion in the microfilter reactor could be controlled simply by regulating the transmembrane pressure drop. This study suggests that a ceramic microfilter reactor can be a desirable alternative to a packed bed of porous particles, especially when an immobilized enzyme has high activity and a low Michaelis constant.

Tough Self-Healing Elastomers by Molecular Enforced Integration of Covalent and Reversible Networks.

Science.gov (United States)

Wu, Jinrong; Cai, Li-Heng; Weitz, David A

2017-10-01

Self-healing polymers crosslinked by solely reversible bonds are intrinsically weaker than common covalently crosslinked networks. Introducing covalent crosslinks into a reversible network would improve mechanical strength. It is challenging, however, to apply this concept to "dry" elastomers, largely because reversible crosslinks such as hydrogen bonds are often polar motifs, whereas covalent crosslinks are nonpolar motifs. These two types of bonds are intrinsically immiscible without cosolvents. Here, we design and fabricate a hybrid polymer network by crosslinking randomly branched polymers carrying motifs that can form both reversible hydrogen bonds and permanent covalent crosslinks. The randomly branched polymer links such two types of bonds and forces them to mix on the molecular level without cosolvents. This enables a hybrid "dry" elastomer that is very tough with fracture energy 13500 Jm -2 comparable to that of natural rubber. Moreover, the elastomer can self-heal at room temperature with a recovered tensile strength 4 MPa, which is 30% of its original value, yet comparable to the pristine strength of existing self-healing polymers. The concept of forcing covalent and reversible bonds to mix at molecular scale to create a homogenous network is quite general and should enable development of tough, self-healing polymers of practical usage. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Immobilization of Isolated Lipase From Moldy Copra (Aspergillus Oryzae

Directory of Open Access Journals (Sweden)

Seniwati Dali

2011-01-01

Full Text Available Enzyme immobilization is a recovery technique that has been studied in several years, using support as a media to help enzyme dissolutions to the reaction substrate. Immobilization method used in this study was adsorption method, using specific lipase from Aspergillus oryzae. Lipase was partially purified from the culture supernatant of Aspergillus oryzae. Enzyme was immobilized by adsorbed on silica gel. Studies on free and immobilized lipase systems for determination of optimum pH, optimum temperature, thermal stability and reusability were carried out. The results showed that free lipase had optimum pH 8,2 and optimum temperature 35 °C while the immobilized lipase had optimum 8,2 and optimum temperature 45 °C. The thermal stability of the immobilized lipase, relative to that of the free lipase, was markedly increased. The immobilized lipase can be reused for at least six times.

Immobile Complex Verbs in Germanic

DEFF Research Database (Denmark)

Vikner, Sten

2005-01-01

the V° requirements or the V* requirements. Haider (1993, p. 62) and Koopman (1995), who also discuss such immobile verbs, only account for verbs with two prefix-like parts (e.g., German uraufführen ‘to perform (a play) for the first time' or Dutch herinvoeren ‘to reintroduce'), not for the more...... frequent type with only one prefix-like part (e.g., German bauchreden/Dutch buikspreken ‘to ventriloquize'). This analysis will try to account not only for the data discussed in Haider (1993) and Koopman (1995) but also for the following: - why immobile verbs include verbs with only one prefix-like part...... are immobile, - why such verbs are not found in Germanic VO-languages such as English and Scandinavian....

The influence of carbon nanotubes on enzyme activity and structure: investigation of different immobilization procedures through enzyme kinetics and circular dichroism studies

International Nuclear Information System (INIS)

Cang-Rong, Jason Teng; Pastorin, Giorgia

2009-01-01

In the last decade, many environmental organizations have devoted their efforts to identifying renewable biosystems, which could provide sustainable fuels and thus enhance energy security. Amidst the myriad of possibilities, some biofuels make use of different types of waste biomasses, and enzymes are often employed to hydrolyze these biomasses and produce sugars that will be subsequently converted into ethanol. In this project, we aimed to bridge nanotechnology and biofuel production: here we report on the activity and structure of the enzyme amyloglucosidase (AMG), physically adsorbed or covalently immobilized onto single-walled carbon nanotubes (SWNTs) and multi-walled carbon nanotubes (MWNTs). In fact, carbon nanotubes (CNTs) present several properties that render them ideal support systems, without the diffusion limitations displayed by porous material and with the advantage of being further functionalizable at their surface. Chemical ligation was achieved both on oxidized nanotubes (via carbodiimide chemistry), as well as on amino-functionalized nanotubes (via periodate-oxidized AMG). Results showed that AMG retained a certain percentage of its specific activity for all enzyme-carbon nanotubes complexes prepared, with the physically adsorbed samples displaying better catalytic efficiency than the covalently immobilized samples. Analysis of the enzyme's structure through circular dichroism (CD) spectroscopy revealed significant structural changes in all samples, the degree of change being consistent with the activity profiles. This study proves that AMG interacts differently with carbon nanotubes depending on the method employed. Due to the higher activity reported by the enzyme physically adsorbed onto CNTs, these samples demonstrated a vast potential for further development. At the same time, the possibility of inducing magnetic properties into CNTs offers the opportunity to easily separate them from the original solution. Hence, substances to which they

Plutonium Immobilization Can Loading Equipment Review

International Nuclear Information System (INIS)

Kriikku, E.; Ward, C.; Stokes, M.; Randall, B.; Steed, J.; Jones, R.; Hamilton, L.

1998-05-01

This report lists the operations required to complete the Can Loading steps on the Pu Immobilization Plant Flow Sheets and evaluates the equipment options to complete each operation. This report recommends the most appropriate equipment to support Plutonium Immobilization Can Loading operations

Characterization of immobilized post-carbohydrate meal salivary α ...

African Journals Online (AJOL)

The effect of experimental parameters like pH, temperature and substrate concentration on the activity of the immobilized post-carbohydrate meal salivary ... of immobilized post-carbohydrate meal salivary α-amylase in this study show that immobilization had no significant effect on the enzyme and compared to kinetic ...

Immobilization of thorium over fibroin by polyacrylonitrile (PAN)

International Nuclear Information System (INIS)

Aslani, M.A.A.; Akyil, S.; Eral, M.

1997-01-01

This report describes a process for immobilization of thorium over fibroin, which was used as a bio-adsorbant, by polyacrylonitrile. The amounts of thorium in aqueous solutions which may be leached in various aqueous ambients were detected by a spectrophotometer. The results show that polyacrylonitrile processes are feasible to immobilize spent fibroins. The leachability of the materials immobilized with polyacrylonitrile can meet the requirements of storage and final disposal. The leachability of thorium ions from immobilized spent fibroin was rather low for 8 months

Metabolic alkalosis during immobilization in monkeys (M. nemestrina)

Science.gov (United States)

Young, D. R.; Yeh, I.; Swenson, R. S.

1983-01-01

The systemic and renal acid-base response of monkeys during ten weeks of immobilization was studied. By three weeks of immobilization, arterial pH and bicarbonate concentrations were elevated (chronic metabolic alkalosis). Net urinary acid excretion increased in immobilized animals. Urinary bicarbonate excretion decreased during the first three weeks of immobilization, and then returned to control levels. Sustained increases in urinary ammonium excretion were seen throughout the time duration of immobilization. Neither potassium depletion nor hypokalemia was observed. Most parameters returned promptly to the normal range during the first week of recovery. Factors tentatively associated with changes in acid-base status of monkeys include contraction of extracellular fluid volume, retention of bicarbonate, increased acid excretion, and possible participation of extrarenal buffers.

Immobilization of ammonia-oxidizing bacteria by polyvinyl alcohol and sodium alginate.

Science.gov (United States)

Dong, Yuwei; Zhang, Yanqiu; Tu, Baojun

Ammonia-oxidizing bacteria were immobilized by polyvinyl alcohol (PVA) and sodium alginate. The immobilization conditions and ammonia oxidation ability of the immobilized bacteria were investigated. The following immobilization conditions were observed to be optimal: PVA, 12%; sodium alginate, 1.1%; calcium chloride, 1.0%; inoculum concentration, 1.3 immobilized balls/mL of immobilized medium; pH, 10; and temperature, 30°C. The immobilized ammonia-oxidizing bacteria exhibited strong ammonia oxidation ability even after being recycled four times. The ammonia nitrogen removal rate of the immobilized ammonia-oxidizing bacteria reached 90.30% under the optimal immobilization conditions. When compared with ammonia-oxidizing bacteria immobilized by sodium alginate alone, the bacteria immobilized by PVA and sodium alginate were superior with respect to pH resistance, the number of reuses, material cost, heat resistance, and ammonia oxidation ability. Copyright © 2017 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

Radiation technology for immobilization of bioactive materials

International Nuclear Information System (INIS)

1988-12-01

Within the framework of the Agency's coordinated research programme on ''Application of Radiation Technology in Immobilization of Bioactive Materials'', the third and final research coordination meeting was held at Beijing University, Beijing, People's Republic of China, 15-18 June 1987. The present publication compiles all presentations made at the meeting. Fundamental processes for the immobilization of enzymes, antibodies, cells and drugs were developed and established using gamma radiation, electron beams and plasma discharge. Applications of various biofunctional components, immobilized by radiation techniques in different processes, were studied. A range of backbone polymers has been examined together with various monomers. Coupling procedures have been developed which are relevant to our particular requirements. Enzymes of various types and characteristics have been immobilized with considerable efficiency. The immobilized biocatalysts have been shown to possess significant activity and retention of activity on storage. There appears to be a high degree of specificity associated with the properties of the immobilised biocatalysts, their activity and the ease of their preparation. Novel additives which lower the total radiation dose in grafting have been discovered and their value in immobilization processes assessed. Potential applications include: medical (diagnostic, therapeutic), and industrial processes (fermentation, bioseparation, etc.). Refs, figs and tabs

Thermal stability of the immobilized fructosyltransferase from Rhodotorula sp

Directory of Open Access Journals (Sweden)

E Aguiar-Oliveira

2011-09-01

Full Text Available The thermal stability of the extracellular fructosyltransferase (FTase from Rhodotorula sp., recovered from cultivation medium by ethanol precipitation and immobilized onto niobium ore, was studied by Arrhenius plot, half - life profile, half - inactivation temperature (T50 and thermodynamic parameters. The Arrhenius plot showed two different behaviors with different deactivation energies (Ead only after immobilization, the transition occurring in the temperature interval between 51 and 52ºC. T50 for the free enzyme was estimated to be around 62ºC and, after immobilization, 66ºC. After 15 minutes at 52ºC, it was also possible to observe enzymatic activation for both the free and immobilized forms, but greater activation was achieved at pH 4.5 with the immobilized enzyme. Between 47 - 51ºC the immobilized enzyme was more stable than the free enzyme, with pH 6.0 being the more stable condition for the immobilized enzyme. However, above 52ºC the free form was more stable.

Application of magnetic nanoparticles in smart enzyme immobilization.

Science.gov (United States)

Vaghari, Hamideh; Jafarizadeh-Malmiri, Hoda; Mohammadlou, Mojgan; Berenjian, Aydin; Anarjan, Navideh; Jafari, Nahideh; Nasiri, Shahin

2016-02-01

Immobilization of enzymes enhances their properties for efficient utilization in industrial processes. Magnetic nanoparticles, due to their high surface area, large surface-to-volume ratio and easy separation under external magnetic fields, are highly valued. Significant progress has been made to develop new catalytic systems that are immobilized onto magnetic nanocarriers. This review provides an overview of recent developments in enzyme immobilization and stabilization protocols using this technology. The current applications of immobilized enzymes based on magnetic nanoparticles are summarized and future growth prospects are discussed. Recommendations are also given for areas of future research.

Temperature Activated Diffusion of Radicals through Ion Implanted Polymers

DEFF Research Database (Denmark)

Wakelin, Edgar A.; Davies, Michael J.; Bilek, Marcela M. M.

2015-01-01

Plasma immersion ion implantation (PIII) is a promising technique for immobilizing biomolecules on the surface of polymers. Radicals generated in a subsurface layer by PIII treatment diffuse throughout the substrate, forming covalent bonds to molecules when they reach the surface. Understanding...... to the surface. The model makes useful predictions for the lifetime over which the surface is sufficiently active to covalently immobilize biomolecules and it can be used to determine radical fluence during biomolecule incubation for a range of storage and incubation temperatures so facilitating selection...

Immobilization of pH-sensitive CdTe Quantum Dots in a Poly(acrylate) Hydrogel for Microfluidic Applications

Science.gov (United States)

Franke, M.; Leubner, S.; Dubavik, A.; George, A.; Savchenko, T.; Pini, C.; Frank, P.; Melnikau, D.; Rakovich, Y.; Gaponik, N.; Eychmüller, A.; Richter, A.

2017-04-01

Microfluidic devices present the basis of modern life sciences and chemical information processing. To control the flow and to allow optical readout, a reliable sensor material that can be easily utilized for microfluidic systems is in demand. Here, we present a new optical readout system for pH sensing based on pH sensitive, photoluminescent glutathione capped cadmium telluride quantum dots that are covalently immobilized in a poly(acrylate) hydrogel. For an applicable pH sensing the generated hybrid material is integrated in a microfluidic sensor chip setup. The hybrid material not only allows in situ readout, but also possesses valve properties due to the swelling behavior of the poly(acrylate) hydrogel. In this work, the swelling property of the hybrid material is utilized in a microfluidic valve seat, where a valve opening process is demonstrated by a fluid flow change and in situ monitored by photoluminescence quenching. This discrete photoluminescence detection (ON/OFF) of the fluid flow change (OFF/ON) enables upcoming chemical information processing.

Ferrocenyl-doped silica nanoparticles as an immobilized affinity support for electrochemical immunoassay of cancer antigen 15-3

International Nuclear Information System (INIS)

Hong Chenglin; Yuan Ruo; Chai Yaqin; Zhuo Ying

2009-01-01

The aim of this study is to elaborate a simple and sensitive electrochemical immunoassay using ferrocenecarboxylic (Fc-COOH)-doped silica nanoparticles (SNPs) as an immobilized affinity support for cancer antigen 15-3 (CA 15-3) detection. The Fc-COOH-doped SNPs with redox-active were prepared by using a water-in-oil microemulsion method. The use of colloidal silica could prevent the leakage of Fc-COOH and were easily modified with trialkoxysilane reagents for covalent conjugation of CA 15-3 antibodies (anti-CA 15-3). The Fc-COOH-doped SNPs were characterized by X-ray photoelectron spectroscopy (XPS) and transmission electron microscopy (TEM). The fabrication process of the electrochemical immunosensor was demonstrated by using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) techniques. Under optimal conditions, the developed immunosensor showed good linearity at the studied concentration range of 2.0-240 U mL -1 with a coefficient 0.9986 and a detection limit of 0.64 U mL -1 at S/N = 3

Ferrocenyl-doped silica nanoparticles as an immobilized affinity support for electrochemical immunoassay of cancer antigen 15-3.

Science.gov (United States)

Hong, Chenglin; Yuan, Ruo; Chai, Yaqin; Zhuo, Ying

2009-02-09

The aim of this study is to elaborate a simple and sensitive electrochemical immunoassay using ferrocenecarboxylic (Fc-COOH)-doped silica nanoparticles (SNPs) as an immobilized affinity support for cancer antigen 15-3 (CA 15-3) detection. The Fc-COOH-doped SNPs with redox-active were prepared by using a water-in-oil microemulsion method. The use of colloidal silica could prevent the leakage of Fc-COOH and were easily modified with trialkoxysilane reagents for covalent conjugation of CA 15-3 antibodies (anti-CA 15-3). The Fc-COOH-doped SNPs were characterized by X-ray photoelectron spectroscopy (XPS) and transmission electron microscopy (TEM). The fabrication process of the electrochemical immunosensor was demonstrated by using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) techniques. Under optimal conditions, the developed immunosensor showed good linearity at the studied concentration range of 2.0-240 UmL(-1) with a coefficient 0.9986 and a detection limit of 0.64 UmL(-1) at S/N=3.