Search results for: laccase immobilization

Authors: Milica B. Veljković, Milica B. Simović, Marija M. Ćorović, Ana D. Milivojević, Anja I. Petrov, Katarina M. Banjanac, Dejan I. Bezbradica

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In recent decades, the scientific community has recognized the growing importance of prebiotics, and therefore, numerous studies are focused on their economic production due to their low presence in natural resources. It has been confirmed that prebiotics is a source of energy for probiotics in the gastrointestinal tract (GIT) and enable their proliferation, consequently leading to the normal functioning of the intestinal microbiota. Also, products of their fermentation are short-chain fatty acids (SCFA), which play a key role in maintaining and improving the health not only of the GIT but also of the whole organism. Among several confirmed prebiotics, fructooligosaccharides (FOS) are considered interesting candidates for use in a wide range of products in the food industry. They are characterized as low-calorie and non-cariogenic substances that represent an adequate sugar substitute and can be considered suitable for use in products intended for diabetics. The subject of this research will be the production of FOS by transforming sucrose using a fructosyltransferase (FTase) present in commercial preparation Pectinex® Ultra SP-L, with special emphasis on the development of adequate FTase immobilization method that would enable selective isolation of the enzyme responsible for the synthesis of FOS from the complex enzymatic mixture. This would lead to considerable enzyme purification and allow its direct incorporation into different sucrose-based products without the fear that the action of the other hydrolytic enzymes may adversely affect the products' functional characteristics. Accordingly, the possibility of selective immobilization of the enzyme using support with primary amino groups, Purolite® A109, which was previously activated and modified using glutaraldehyde (GA), was investigated. In the initial phase of the research, the effects of individual immobilization parameters such as pH, enzyme concentration, and immobilization time were investigated to optimize the process using support chemically activated with 15% and 0.5% GA to form dimers and monomers, respectively. It was determined that highly active immobilized preparations (371.8 IU/g of support - dimer and 213.8 IU/g of support – monomer) were achieved under acidic conditions (pH 4) provided that an enzyme concentration was 50 mg/g of support after 7 h and 3 h, respectively. Bearing in mind the obtained results of the expressed activity, it is noticeable that the formation of dimers showed higher reactivity compared to the form of monomers. Also, in the case of support modification using 15% GA, the value of the ratio of FTase and pectinase (as dominant enzyme mixture component) activity immobilization yields was 16.45, indicating the high feasibility of selective immobilization of FTase on modified polystyrene resin. After obtaining immobilized preparations of satisfactory features, they were tested in a reaction of FOS synthesis under determined optimal conditions. The maximum FOS yields of approximately 50% of total carbohydrates in the reaction mixture were recorded after 21 h. Finally, it can be concluded that the examined immobilization method yielded highly active, stable and, more importantly, refined enzyme preparation that can be further utilized on a larger scale for the development of continual processes for FOS synthesis, as well as for modification of different sucrose-based mediums.

Keywords: chemical modification, fructooligosaccharides, glutaraldehyde, immobilization of fructosyltransferase

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Authors: Homa Torabizadeh, Mohaddeseh Mikani

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Inulinase from Aspergillus niger was covalently immobilized on magnetic nanoparticles (MNPs/Fe₃O₄) covered with soy protein isolate (SPI/Fe₃O₄) functionalized by bovine serum albumin (BSA) nanoparticles. MNPs are promising enzyme carriers because they separate easily under external magnetic fields and have enhanced immobilized enzyme reusability. As MNPs aggregate simply, surface coating strategy was employed. SPI functionalized by BSA was a suitable candidate for nanomagnetite coating due to its superior biocompatibility and hydrophilicity. Fe₃O₄@SPI-BSA nanoparticles were synthesized as a novel carrier with narrow particle size distribution. Step by step fabrication monitoring of Fe₃O₄@SPI-BSA nanoparticles was performed using field emission scanning electron microscopy and dynamic light scattering. The results illustrated that nanomagnetite with the spherical morphology was well monodispersed with the diameter of about 35 nm. The average size of the SPI-BSA nanoparticles was 80 to 90 nm, and their zeta potential was around −34 mV. Finally, the mean diameter of fabricated Fe₃O₄@SPI-BSA NPs was less than 120 nm. Inulinase enzyme from Aspergillus niger was covalently immobilized through gluteraldehyde on Fe₃O₄@SPI-BSA nanoparticles successfully. Fourier transform infrared spectra and field emission scanning electron microscopy images provided sufficient proof for the enzyme immobilization on the nanoparticles with 80% enzyme loading.

Keywords: high fructose syrup, inulinase immobilization, functionalized magnetic nanoparticles, soy protein isolate

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Authors: Bongmun Kang, Kagnari Yamakawa, Yoshihisa Hagihara, Yuji Ito, Michimasa Kishimoto, Yoichi Kumada

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The PMMA-tag was genetically fused with the C-terminal region of VHH molecules. This antibody, VHH, is known as a single-chain domain, which is devoid of light chains. The PMMA-tag, which could affect the isoelectric point (pI) changeable with a charge of amino acid in VHHs were closely related to the solubility of VHH molecules during refolding. The genetic fusion of PMMA-tag to C-terminal region of VHHs significantly affects the recovery of their soluble protein during refolding by 50 mM TAPS at pH 8.5. It could be refolded with a recovery of more than 95% by dialysis at pH 8.5. A marked difference in the antigen-binding activities in the adsorption state was significantly high in VHH-PM compared to the wild type of VHH. There are approximately 8-fold differences in the antigen-binding activities in the adsorption state between VHH-PM and VHH.

Keywords: VHH, PMMA-tag, isoelectric point, pH, Solubility, refolding, immobilization, ELISA

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Authors: Gajanan V. Mane, Rashmi More, Mahesh S. Sonawane, Tushar Lodha, Rohit Sharma

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The diversity of fungi isolated from two different termite species viz., Odontoterms assmuthi and O. abesus was investigated by dilution- plate method, combined with morphological characteristics and sequencing of internal transcribed spacer region. In total, ninety-six fungi were isolated and purified, out of which 69 isolates were obtained from O. assmuthi belonging to 18 genera and 31 species, whereas 27 isolates were obtained from O. abesus belonging to 15 genera and 17 species. The fungal strains were screened for laccase, amylase, cellulase and pectinase enzymes production. Twenty-seven strains were positive for laccase, 59 strains were positive for amylase, 71 strains were positive for cellulase and 72 strains were positive for pectinase enzymes. The antimicrobial activities of the isolated fungi were tested by the dual plate culture method against standard pathogens. Bioactive secondary metabolites were identified by HPLC and LCMS. Four isolates viz., Penicillium goetzii MG 57, Epicoccum sp. MG 39, Penicillium tanzanicum MG 30, Aspergillus polyporicola MG 54, showed positive antimicrobial activity against standard pathogens, Streptococcus pneumonia MCC 2425, Staphylococcus aureus MCC 2408, Pseudomonas aeruginosa MCC 2080, Escherichia coli MCC 2412, Enterococcus faecalis MCC 2409, Klebsiella pneumonia MCC 2451, Micrococcus luteus MCC 2155 and Candida albicans MCC 1151. In conclusion, the study showed that the insect gut harbor fungal diversity, which is futuristic with biotechnological potential and could be a good source of enzymes and antibiotics.

Keywords: termites, fungi, its, enzyme, antimicrobial activity

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Authors: Antonella Cartoni, Mattea Carmen Castrovilli

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A biosensor for lactate detection has been developed using an environmentally friendly approach. The biosensor is based on lactate oxidase (LOX) and has remarkable capabilities for reuse and storage at room temperature. The manufacturing technique employed is ambient electrospray deposition (ESD), which enables efficient and sustainable immobilization of the LOX enzyme on a cost-effective com-mercial screen-printed Prussian blue/carbon electrode (PB/C-SPE). The study demonstrates that the ESD technology allows the biosensor to be stored at ambient pressure and temperature for extended periods without affecting the enzymatic activity. The biosensor can be stored for up to 90 days without requiring specific storage conditions, and it can be reused for up to 24 measurements on both freshly prepared electrodes and electrodes that are three months old. The LOX-based biosensor exhibits a lin-ear range of lactate detection between 0.1 and 1 mM, with a limit of detection of 0.07±0.02 mM. Ad-ditionally, it does not exhibit any memory effects. The immobilization process does not involve the use of entrapment matrices or hazardous chemicals, making it environmentally sustainable and non-toxic compared to current methods. Furthermore, the application of a electrospray deposition cycle on previously used biosensors rejuvenates their performance, making them comparable to freshly made biosensors. This highlights the excellent recycling potential of the technique, eliminating the waste as-sociated with disposable devices.

Keywords: green friendly, reuse, storage performance, immobilization, matrix-free, electrospray deposition, biosensor, lactate oxidase, enzyme

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

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

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

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

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

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

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Authors: A. G. Pershina, P. S. Postnikov, M. E. Trusova, D. O. Burlakova, A. E. Sazonov

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Invention of magnetic-fluorescent nanocomposites is a rapidly developing area of research. The magnetic-fluorescent nanocomposite attractiveness is connected with the ability of simultaneous management and control of such nanocomposites by two independent methods based on different physical principles. These nanocomposites are applied for the solution of various essential scientific and experimental biomedical problems. The aim of this research is development of principle approach to nanobiohybrid structures with magnetic and fluorescent properties design. The surface of carbon-coated iron nanoparticles (Fe@C) were covalently modified by 4-carboxy benzenediazonium tosylate. Recombinant fluorescent protein TagGFP2 (Eurogen) was obtained in E. coli (Rosetta DE3) by standard laboratory techniques. Immobilization of TagGFP2 on the nanoparticles surface was provided by the carbodiimide activation. The amount of COOH-groups on the nanoparticle surface was estimated by elemental analysis (Elementar Vario Macro) and TGA-analysis (SDT Q600, TA Instruments. Obtained nanocomposites were analyzed by FTIR spectroscopy (Nicolet Thermo 5700) and fluorescence microscopy (AxioImager M1, Carl Zeiss). Amount of the protein immobilized on the modified nanoparticle surface was determined by fluorimetry (Cary Eclipse) and spectrophotometry (Unico 2800) with the help of preliminary obtained calibration plots. In the FTIR spectra of modified nanoparticles the adsorption band of –COOH group around 1700 cm-1 and bands in the region of 450-850 cm-1 caused by bending vibrations of benzene ring were observed. The calculated quantity of active groups on the surface was equal to 0,1 mmol/g of material. The carbodiimide activation of COOH-groups on nanoparticles surface results to covalent immobilization of TagGFP2 fluorescent protein (0.2 nmol/mg). The success of immobilization was proved by FTIR spectroscopy. Protein characteristic adsorption bands in the region of 1500-1600 cm-1 (amide I) were presented in the FTIR spectrum of nanocomposite. The fluorescence microscopy analysis shows that Fe@C-TagGFP2 nanocomposite possesses fluorescence properties. This fact confirms that TagGFP2 protein retains its conformation due to immobilization on nanoparticles surface. Magnetic-fluorescent nanocomposite was obtained as a result of unique design solution implementation – the fluorescent protein molecules were fixed to the surface of superparamagnetic carbon-coated iron nanoparticles using original diazonium salts.

Keywords: carbon-coated iron nanoparticles, diazonium salts, fluorescent protein, immobilization

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Authors: Angelika-Ioanna Gialleli, Gousi Mantha, Maria Kanellaki, Bekatorou Argyro, Athanasios Koutinas

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In this study, a new brewing technology with dry raw materials is proposed with potential application in home brewing. Bio catalysts were prepared by immobilization of the psychrotolerant yeast strain Saccharomyces cerevisiae AXAZ-1 on tubular cellulose. Both the word and the biocatalysts were freeze-dried without any cryoprotectants and used for low temperature brewing. The combination of immobilization and freeze-drying techniques was applied successfully, giving a potential for supplying breweries with preserved and ready-to-use immobilized cells. The effect of wort sugar concentration (7°, 8.5°, 10°Be), temperature (2, 5, 7° C) and carrier concentration (5, 10, 20 g/L) on fermentation kinetics and final product quality (volatiles, colour, polyphenols, bitterness) was assessed. The same procedure was repeated with free cells for comparison of the results. The results for immobilized cells were better compared to free cells regarding fermentation kinetics and organoleptic characteristics.

Keywords: brewing, tubular cellulose, low temperature, biocatalyst

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Authors: Afsheen Aman, Zainab Bibi, Shah Ali Ul Qader

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Introduction: Xylan is a heteropolysaccharide composed of xylose monomers linked together through 1,4 linkages within a complex xylan network. Owing to wide applications of xylan hydrolytic products (xylose, xylobiose and xylooligosaccharide) the researchers are focusing towards the development of various strategies for efficient xylan degradation. One of the most important strategies focused is the use of heat tolerant biocatalysts which acts as strong and specific cleaving agents. Therefore, the exploration of microbial pool from extremely diversified ecosystem is considerably vital. Microbial populations from extreme habitats are keenly explored for the isolation of thermophilic entities. These thermozymes usually demonstrate fast hydrolytic rate, can produce high yields of product and are less prone to microbial contamination. Another possibility of degrading xylan continuously is the use of immobilization technique. The current work is an effort to merge both the positive aspects of thermozyme and immobilization technique. Methodology: Geobacillus stearothermophilus was isolated from soil sample collected near the blast furnace site. This thermophile is capable of producing thermostable endo-β-1,4-xylanase which cleaves xylan effectively. In the current study, this thermozyme was immobilized within a synthetic and a non-synthetic matrice for continuous production of metabolites using entrapment technique. The kinetic parameters of the free and immobilized enzyme were studied. For this purpose calcium alginate and polyacrylamide beads were prepared. Results: For the synthesis of immobilized beads, sodium alginate (40.0 gL-1) and calcium chloride (0.4 M) was used amalgamated. The temperature (50°C) and pH (7.0) optima of immobilized enzyme remained same for xylan hydrolysis however, the enzyme-substrate catalytic reaction time raised from 5.0 to 30.0 minutes as compared to free counterpart. Diffusion limit of high molecular weight xylan (corncob) caused a decline in Vmax of immobilized enzyme from 4773 to 203.7 U min-1 whereas, Km value increased from 0.5074 to 0.5722 mg ml-1 with reference to free enzyme. Immobilized endo-β-1,4-xylanase showed its stability at high temperatures as compared to free enzyme. It retained 18% and 9% residual activity at 70°C and 80°C, respectively whereas; free enzyme completely lost its activity at both temperatures. The Immobilized thermozyme displayed sufficient recycling efficiency and can be reused up to five reaction cycles, indicating that this enzyme can be a plausible candidate in paper processing industry. Conclusion: This thermozyme showed better immobilization yield and operational stability with the purpose of hydrolyzing the high molecular weight xylan. However, the enzyme immobilization properties can be improved further by immobilizing it on different supports for industrial purpose.

Keywords: immobilization, reusability, thermozymes, xylanase

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Authors: Mohamed Elbehery

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Historical textiles in the burial environment or in museums are exposed to many types of stains and dirt that are associated with historical textiles by multiple chemical bonds that cause damage to historical textiles. The cleaning process must be carried out with great care, with no irreversible damage, and sediments removed without affecting the original material of the surface being cleaned. Science and technology continue to provide innovative systems in the bio-cleaning process (using pure enzymes) of historical textiles and artistic surfaces. Lipase and α-amylase were immobilized on nanoparticles of alginate/κ-carrageenan nanoparticle complex and used in historical textiles cleaning. Preparation of nanoparticles, activation, and enzymes immobilization were characterized. Optimization of loading time and units of the two enzymes were done. It was found that, the optimum time and units of amylase were 4 hrs and 25U, respectively. While, the optimum time and units of lipase were 3 hrs and 15U, respectively. The methods used to examine the fibers using a scanning electron microscope equipped with an X-ray energy dispersal unit: SEM with EDX unit.

Keywords: nanoparticles, enzymes, immobilization, textiles

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Authors: Sasan Talebnezhad, Saeed Pormahdian, Naghi Assali

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Homogeneous α-diimine nickel (II) catalyst complexes, with and without amino para-aryl position functionality, were synthesized. These complexes were immobilized on carboxyl, hydroxyl, and acyl chloride functionalized multi-walled carbon nanotubes to form five novel heterogeneous α-diiminonickel catalysts. Immobilization was performed by covalent or electrostatic bonding via methylaluminoxane (MAO) linker or amide linkage. Both the nature of α-diimine ligands and the kind of interaction between anchored catalyst complexes and multi-walled carbon nanotube surface influenced the catalytic performance, microstructure, and morphology of obtained polyethylenes. The catalyst prepared by amide bonding showed lowest relative weight loss in thermogravimetry analysis and highest activities up to 5863 gr PE mmol-1Ni.hr-1. This catalyst produced polyethylene with dense botryoidal morphology.

Keywords: α-diimine nickel (II) complexes, immobilization, multi-walled carbon nanotubes, ethylene polymerization

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Authors: Sasan Talebnezhad, Saeed Pourmahdian, Naghi Assali

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Homogeneous α-diimine nickel (II) catalyst complexes, with and without amino para-aryl position functionality, were synthesized. These complexes were immobilized on carboxyl, hydroxyl and acyl chloride functionalized multi-walled carbon nanotubes to form five novel heterogeneous α diiminonickel catalysts. Immobilization was performed by covalent or electrostatic bonding via methylaluminoxane (MAO) linker or amide linkage. Both the nature of α-diimine ligands and the kind of interaction between anchored catalyst complexes and multi-walled carbon nanotube surface influenced the catalytic performance, microstructure, and morphology of obtained polyethylenes. The catalyst prepared by amide bonding showed lowest relative weight loss in thermogravimetry analysis and highest activities up to 5863 gr PE mmol-1Ni.hr-1. This catalyst produced polyethylene with dense botryoidal morphology.

Keywords: α-diimine nickel (II) complexes, immobilization, multi-walled carbon nanotubes, ethylene polymerization

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Authors: Carolyn Palma, Andrea Carvajal, Luisa Sepúlveda, Jenifer Cavieres, Ricardo Kalm

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Trace organic compounds, specifically pharmaceutical ones, due to their increasing usage have been detected in various water bodies, endangering the ecosystems. Nowadays the treatment for these pollutants leans towards the application of hybrid technologies. This study is focused on the application of a simultaneous adsorption and biodegradation system for pharmaceutical removal using commercial granulated activated carbon (AC), which is the adsorbent agent after being chemically activated with HCl 1M, called functionalized activated carbon (FAC). Oxidative enzyme Laccase was produced from Trametes versicolor. To immobilize the enzymes into the FAC surface, the enzyme was contacted with the support at a rate of 10 mg protein/ mg of FAC at pH 7, at 4°C of temperature and gentle agitation, allowing the production of supports that had 20%, 50%, and 80% of the FAC surface free of the enzyme, called EFAC 20, EFAC 50 and EFAC 80, respectively. A factorial experiment (22) was carried out, with three central replica points, considering as variables: free surface for adsorption (80%, 50% and 20%) and the concentration of pharmaceutical compounds. (50, 125 and 200 mg L⁻¹). This experiment was designed to study the behavior of these supports exposed to ibuprofen (IBU) and acetaminophen (APH). All experimental procedures were carried out at room temperature, keeping a pH level of 7 and a stirring speed of 150 rpm. Supports containing 80% of the free surface (EFAC80) after 216 h of exposure show the best results for pharmaceutical removal at 50 and 200 mg L⁻¹. For APH, there is a 6% variation in the adsorption capacity for both 50 and 200 mg L⁻¹. However, for IBU, these variations were 2% and 1% for concentrations of 50 and 200 mg L⁻¹, respectively. This study shows the importance of not only removing the pollutant but also degrading it. As shown in the results of all cases in the presence of the enzyme, the process allowed to rise the removal capacity, implying that active sites are emptied gradually because of this degradation, enabling these sites to keep interacting successively.

Keywords: activated carbon, adsorption, enzymatic degradation, trace organic compound

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Authors: Joanna Cabaj, Sylwia Baluta, Karol Malecha, Kamila Drzozga

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Catecholamines are the principal neurotransmitters that mediate a variety of the central nervous system functions, such as motor control, cognition, emotion, memory processing, and endocrine modulation. Dysfunctions in catecholamine neurotransmission are induced in some neurologic and neuropsychiatric diseases. Changeable neurotransmitters level in biological fluids can be a marker of several neurological disorders. Because of its significance in analytical techniques and diagnostics, sensitive and selective detection of neurotransmitters is increasingly attracting a lot of attention in different areas of bio-analysis or biomedical research. Recently, fluorescent techniques for detection of catecholamines have attracted interests due to their reasonable cost, convenient control, as well as maneuverability in biological environments. Nevertheless, with the observed need for a sensitive and selective catecholamines sensor, the development of a convenient method for this neurotransmitter is still at its basic level. The manipulation of nanostructured materials in conjunction with biological molecules has led to the development of a new class of hybrid modified biosensors in which both enhancement of charge transport and biological activity preservation may be obtained. Immobilization of biomaterials on electrode surfaces is the crucial step in fabricating electrochemical as well as optical biosensors and bioelectronic devices. Continuing systematic investigation in the manufacturing of enzyme–conducting sensitive systems, here is presented a convenient fluorescence sensing strategy for catecholamines detection based on FRET (fluorescence resonance energy transfer) phenomena observed for, i.e., complexes of Fe²⁺ and epinephrine. The biosensor was constructed using low temperature co-fired ceramics technology (LTCC). This sensing system used the catalytical oxidation of catecholamines and quench of the strong luminescence of obtained complexes due to FRET. The detection process was based on the oxidation of substrate in the presence of the enzyme–laccase/tyrosinase.

Keywords: biosensor, conducting polymer, enzyme, FRET, LTCC

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Authors: M. Cynthya, V. Prabhawathi, D. Mukesh

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Food contamination occurs during post process handling. This leads to spoilage and growth of pathogenic microorganisms in the food, thereby reducing its shelf life or spreading of food borne diseases. Several methods are tried and one of which is use of antimicrobial packaging. Here, papain, a protease enzyme, is covalently immobilized with the help of glutarldehyde on polyurethane and used as a food wrap to protect food from microbial contamination. Covalent immobilization of papain was achieved at a pH of 7.4; temperature of 4°C; glutaraldehyde concentration of 0.5%; incubation time of 24 h; and 50 mg of papain. The formation of -C=N- observed in the Fourier transform infrared spectrum confirmed the immobilization of the enzyme on the polymer. Immobilized enzyme retained higher activity than the native free enzyme. The efficacy of this was studied by wrapping it over S. aureus contaminated cottage cheese (paneer) and cheese and stored at a temperature of 4°C for 7 days. The modified film reduced the bacterial contamination by eight folds when compared to the bare film. FTIR also indicates reduction in lipids, sugars and proteins in the biofilm.

Keywords: cheese, papain, polyurethane, Staphylococcus aureus

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Authors: Md. Shoffikul Islam, Hongqing Hu

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Reducing trace elements' mobility and bioavailability through amendment addition, especially biochar (BC), is a cost-effective and efficient method to address their toxicity in the soil environment. However, the low molecular weight organic acids (LMWOAs) in the rhizosphere could affect BC's efficiency to stabilize trace metals as the LMWOAs could either mobilize or fix metals in the soils. Therefore, understanding the BC's and LMWOAs' interaction mechanisms on metals stabilization in the rhizosphere is crucial. The present study explored the impact of BC derived from rice husk and citric acid (CA) and the combination of BC and CA on the redistribution of cadmium (Cd), lead (Pb), and zinc (Zn) among their geochemical forms through incubation experiment. The changes of zeta potential and X-ray diffraction (XRD) pattern of BC and BC-amended soils to investigate the probable mechanisms of trace elements' immobilization by BC under the CA attack were also examined. The rice husk BC at 5% (w/w) was mixed with the air-dry soil (an Anthrosols) contaminated with Cd, Pb, and Zn in the plastic pot. The 2, 5, 10, and 20 mM kg-1 (w/v) of CA were added separately into the pot. All the ingredients were mixed thoroughly with the soil. A control (CK) treatment was also prepared without BC and CA addition. After 7, 15, and 60 days of incubation with 60% (w/v) moisture level at 25 °C, the incubated soils were determined for pH and EC and were sequentially extracted to assess the metals' transformation in soil. The electronegative charges and XRD peaks of BC and BC-amended soils were also measured. Compared to CK, the application of BC, low level of CA (2 mM kg-1 soil) (CA2), and BC plus the low concentration of CA (BC-CA2) considerably declined the acid-soluble Cd, Pb, and Zn in which BC-CA2 was found to be the most effective treatment. The reversed trends were observed concerning the high levels of CA (>5-20 mM kg-1 soil) and the BC plus high concentrations of CA treatments. BC-CA2 changed the highest amounts of acid-soluble and reducible metals to the oxidizable and residual forms with time. The most increased electronegative charges of BC-CA2 indicate its (BC-CA2) highest Cd, Pb, and Zn immobilizing efficiency, probably through metals adsorption and fixation with the negative charge sites. The XRD study revealed the presence of P, O, CO32-, and Cl1- in BC, which might be responsible for the precipitation of CdCO3, pyromorphite, and hopeite in the case of Cd, Pb, and Zn immobilization, respectively. The findings depicted that the low concentration of CA increased metals' stabilization, whereas the high levels of CA enhanced their mobilization. The BC-CA2 emerged as the best amendment among treatments for metals stabilization in contaminated soils.

Keywords: Biochar, citric acid, immobilization, trace elements contaminated soil

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Authors: Md. Shoffikul Islam, Hongqing Hu

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Immobilizing trace elements by reducing their mobility and bioavailability through amendment application, especially biochar (BC), is a cost-effective and efficient method to address their toxicity in the soil environment. However, the low molecular weight organic acids (LMWOAs) in the rhizosphere could affect BC's efficiency to immobilize trace metals as the LMWOAs could either mobilize or fix metals in the soils. Therefore, understanding the BC's and LMWOAs' interaction mechanisms on metals stabilization in the rhizosphere is crucial. The present study examined the impact of BC derived from rice husk, tartaric acid (TA), and oxalic acid (OA), and the combination of BC and TA/OA on the changes of cadmium (Cd), lead (Pb), and zinc (Zn) among their geochemical forms through incubation experiment. The changes of zeta potential and X-ray diffraction (XRD) pattern of BC and BC-amended soils to investigate the probable mechanisms of trace elements' immobilization by BC under the attacks of TA and OA were also examined. The rice husk BC at 5% (w/w) was mixed with the air-dry soil (an Anthrosols) contaminated with Cd, Pb, and Zn in the plastic pot. The TA and OA each at 2, 5, 10, and 20 mM kg-1 (w/v) were added separately into the pot. All the ingredients were mixed thoroughly with the soil. A control (CK) treatment was also prepared without BC, TA, and OA addition. After 7, 15, and 60 days of incubation with 60% (w/v) moisture level at 25 °C, the incubated soils were determined for pH and EC and were sequentially extracted to assess the metals' transformation in soil. The electronegative charges and XRD peaks of BC and BC-amended soils were also measured. The BC, low level of TA (2 mM kg-1 soil), and BC plus the low concentration of TA (BC-TA2) addition considerably declined the acid-soluble Cd, Pb, and Zn in which BC-TA2 was found to be the most effective treatment. The trends were reversed concerning the high levels of TA (>5-20 mM kg-1 soil), all levels of OA (2-20 mM kg-1 soil), and the BC plus high levels of TA/OA treatments. BC-TA2 changed the highest amounts of acid-soluble and reducible metals to the oxidizable and residual fractions with time. The most increased electronegative charges of BC-TA2 indicate its (BC-TA2) highest metals' immobilizing efficiency, probably through metals adsorption and fixation with the negative charge sites. The XRD study revealed the presence of P, O, CO32-, and Cl1- in BC, which might be responsible for the precipitation of CdCO3, pyromorphite, and hopeite concerning Cd, Pb, and Zn immobilization, respectively. The findings demonstrated that the low level of TA increased metals immobilization, while the high levels of TA and all levels of OA enhanced their mobilization. The BC-TA2 was the best treatment in stabilizing metals in soil.

Keywords: biochar, immobilization, low molecular weight organic acids, trace elements contaminated soil

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Authors: Mirjana Petronijević, Sanja Panić, Aleksandra Cvetanović, Branko Kordić, Nenad Grba

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Enzyme peroxidases are biological catalysts and play a major role in phenolic wastewater treatments and other environmental applications. The most studied species from the peroxidases family is horseradish peroxidase (HRP). In environmental processes, HRP could be used in its free or immobilized form. Enzyme immobilization onto solid support is performed to improve the enzyme properties, prolong its lifespan and operational stability and allow its reuse in industrial applications. One of the enzyme supports of a newer generation is magnetic particles (MPs). Fe₃O₄ MPs are the most widely pursued immobilization of enzymes owing to their remarkable advantages of biocompatibility and non-toxicity. Also, MPs can be easily separated and recovered from the water by applying an external magnetic field. On the other hand, metals and metal oxides are not suitable for the covalent binding of enzymes, so it is necessary to perform their surface modification. Fe₃O₄ MPs functionalization could be performed during the process of their synthesis if it takes place in the presence of plant extracts. Extracts of plant material, such as wild plants, herbs, even waste materials of the food and agricultural industry (bark, shell, leaves, peel), are rich in various bioactive components such as polyphenols, flavonoids, sugars, etc. When the synthesis of magnetite is performed in the presence of plant extracts, bioactive components are incorporated into the surface of the magnetite, thereby affecting its functionalization. In this paper, the suitability of bio-magnetite as solid support for covalent immobilization of HRP across glutaraldehyde was examined. The activity of immobilized HRP at different pH values (4-9) and temperatures (20-80°C) and reusability were examined. Bio-MP was synthesized by co-precipitation method from Fe(II) and Fe(III) sulfate salts in the presence of water extract of the Allium cepa peel. The water extract showed 81% of antiradical potential (according to DPPH assay), which is connected with the high content of polyphenols. According to the FTIR analysis, the bio-magnetite contains oxygen functional groups (-OH, -COOH, C=O) suitable for binding to glutaraldehyde, after which the enzyme is covalently immobilized. The immobilized enzyme showed high activity at ambient temperature and pH 7 (30 U/g) and retained ≥ 80% of its activity at a wide range of pH (5-8) and temperature (20-50°C). The HRP immobilized onto bio-MPs showed remarkable stability towards temperature and pH variations compared to the free enzyme form. On the other hand, immobilized HRP showed low reusability after the first washing cycle enzyme retains 50% of its activity, while after the third washing cycle retains only 22%.

Keywords: bio-magnetite, enzyme immobilization, water extracts, environmental protection

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Authors: Leila Mosafa, Majid Moghadam, Mohammad Shahedi

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

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

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Authors: Srinivasa Reddy Mallampati, Chi-Hyeon Lee, Nguyen Thi Thanh Truc, Byeong-Kyu Lee

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In the present study, feasibility of the selective surface hydrophilization of polyvinyl chloride (PVC) by microwave treatment was evaluated to facilitate the separation from automotive shredder residue (ASR), by the froth flotation. The combination of 60 sec microwave treatment with PAC, a sharp and significant decrease about 16.5° contact angle of PVC was observed in ASR plastic compared with other plastics. The microwave treatment with the addition of PAC resulted in a synergetic effect for the froth flotation, which may be a result of the 90% selective separation of PVC from ASR plastics, with 82% purity. While, simple mixing with a nanometallic Ca/CaO/PO4 dispersion mixture immobilized 95-100% of heavy metals in ASR soil/residues. The quantity of heavy metals leached from thermal residues after treatment by nanometallic Ca/CaO/PO4 was lower than the Korean standard regulatory limit for hazardous waste landfills. Microwave treatment can be a simple and effective method for PVC separation from ASR plastics.

Keywords: automotive shredder residue, chlorinated plastics, hazardous waste, heavy metals, immobilization, separation

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

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

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

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Authors: Mohamed E. Hassan

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Through numerous chemical linkages, historical textiles in burial contexts or in museums are exposed to many different forms of stains and filth. The cleaning procedure must be carried out carefully without causing any irreparable harm, and sediments must be removed without damaging the surface's original material. Science and technology continue to develop novel methods for cleaning historical textiles and artistic surfaces biologically (using enzymes). Lipase and α-amylase were immobilized on nanoparticles of alginate/tea waste nanoparticle complex and used in historical textile cleaning. The preparation of nanoparticles, activation, and enzyme immobilization were characterized. Optimization of loading times and units of the two enzymes was done. It was found that the optimum time and units of amylase were 3 hours and 30 U, respectively. While the optimum time and units of lipase were 2.5 hours and 20 U, respectively, FT-IR and TGA instruments were used in proving the preparation of nanoparticles and the immobilization process. SEM was used to examine the fibres before and after treatment. In conclusion, a new carrier was prepared from alginate/Tea waste and optimized to be used in the restoration and conservation of historical textiles using immobilized lipase and α-amylase.

Keywords: alginate/tea waste, nanoparticles, immobilized enzymes, historical textiles

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Authors: Maryam Samani. Ahmad Golchin. Hosseinali Alikkani. Ahmad Baybordi

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Natural diatomite was modified by grinding and acid treatment to increase surface area and to decrease the impurities. Surface area and pore volume of the modified diatomite were 67.45 m² g-1 and 0.105 cm³ g-¹ respectively, and used to immobilize Pb, Zn and Cu in an urban soil. The modified diatomite was added to soil samples at the rates of 2.5, 5, 7.5 and 10% and the samples incubated for 60 days. The addition of modified diatomite increased SSA of the soil. The SSAs of soils with 2.5, 5.0, 7.5 and 10% modified diatomite were 20.82, 22.02, 23.21 and 24.41 m² g-¹ respectively. Increasing the SSAs of the soils by the application of modified diatomite reduced the DTPA extractable concentrations of heavy metals compared with un-amendment control. The concentration of Pb, Zn and Cu were reduced by 91.1%, 82% and 91.1% respectively. Modified diatomite reduced the concentration of Exchangeable and Carbonate bounded species of Pb, Zn and Cu, compared with the control. Also significantly increased the concentration of Fe Mn- OX (Fe-Mn Oxides) and OM (Organic Matter) bound and Res (Residual) fraction. Modified diatomite increased the urease, dehydrogenase and alkaline phosphatase activity by 52%, 57% and 56.6% respectively.

Keywords: modified diatomite, chemical specifications, specific surface area, enzyme activity, immobilization, heavy metal, soil remediation

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Authors: Blair Arthur Agero Jr., Lucia Garcia Heras

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The triangular fibrocartilage complex (TFCC) is one of the crucial stabilizing ligaments of the wrist. The TFCC is also subject to excessive stress amongst performance athletes and enthusiasts. The excessive loading of the TFCC may lead to a partial or complete rupture that requires surgery. The recovery from an open TFCC surgical repair may take several months. Immobilization of the repaired wrist for a given period is part of all the current protocols in the post-surgical treatment. The immobilization to prevent the rotation of the forearm can last from six weeks to eight weeks with the wrist held in a neutral position. In all protocols reviewed, the pronosupination is only initiated between the 6th week and 8th week or even later after the cast is removed. The prolonged immobilization can cause stiffness of the wrist and hand. Furthermore, the entire period of post-surgical hand therapy has its economic impact, especially for performing athletes. However, delayed mobilization, specifically rotation of the wrist, is necessary to allow ligament healing. This study aims to report the effects of early mobilization of the wrist in athletes who had an open surgical repair of the TFCC. The surgery was done by the co-author, and the hand therapy was implemented by the main author. The cases documented spans from 2014 to 2019 and were all performed in Dubai, United Arab Emirates. All selected participants in this case study were provided with a follow-up questionnaire to ascertain their current condition since their surgery. The respondents reported high satisfaction in the results of their treatment and have verified zero re-rupture of their TFCC despite mobilizing and rotating the wrist at the third-week post-surgery during their hand therapy. There is also a negligible number of respondents who reported a limitation in their ranges of pronosupination. This case study suggests that early mobilization of the wrist after an open TFCC surgical repair can be more beneficial to the patient as opposed to the traditional treatment of prolonged immobilization. However, it should be considered that the patients selected in this case study are professional performance athletes and advanced fitness enthusiasts. Athletes are known to withstand vigorous physical stress in their training that may correlate to their ability to better cope with the progressive stress that was implemented during their hand therapy. Nevertheless, this approach has its merits, and application of it may be adjusted for patients with a similar injury and surgical procedure.

Keywords: hand therapy, performance athlete, TFCC repair, wrist ligament

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Authors: Zhanara B. Suleimenova, Zhazira K. Saduyeva

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Filamentous fungi are major producers of enzymes that have important applications in the food and beverage industries. The overall objective of this research is a strain improvement technology for efficient industrial enzymes production. The new way of filamentous fungi cultivation method has been developed. Such technology prolong producers’ cultivation period up to 60 days and create the opportunity to obtain enzymes repeatedly in every 2-3 days of fungal cultivation. This method is based on immobilizing enzymes producers with solid support in submerged conditions of growth. Immobilizing has a range of advantages: Decreasing the price of the final product, absence of foreign substances, controlled process of enzyme-genesis, ability of various enzymes simultaneous production, etc. Design of proposed technology gives the opportunity to increase the activity of immobilized cells culture filtrate comparing to free cells, growing in periodic culture conditions. Thus, proposed research focuses on new, more versatile, microorganisms capable of squeezing more end-products as well as proposed cultivation technology led to increased enzymatic productivity by several times.

Keywords: filamentous fungi, immobilization, industrial enzymes production, strain improvement

Procedia PDF Downloads 338

Authors: Srinivasa Reddy Mallampati, Byoung Ho Lee, Yoshiharu Mitoma, Simion Cristian

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In recent years, environmental nanotechnology has risen to the forefront and the new properties and enhanced reactivates offered by nanomaterial may offer a new, low-cost paradigm to solving complex environmental pollution problems. This study assessed the synthesis and application of multi-functioned nano-size metallic calcium (nMC) composite for detoxification of hazardous inorganic (heavy metals (HMs)/organic chlorinated/brominated compound (CBCs) contaminants in automobile shredder residue (ASR). ASR residues ball milled with nMC composite can achieve about 90-100% of HMs immobilization and CBCs decomposition. The results highlight the low quantity of HMs leached from ASR residues after treatment with nMC, which was found to be lower than the standard regulatory limit for hazardous waste landfills. The use of nMC composite in a mechanochemical process to treat hazardous ASR (dry conditions) is a simple and innovative approach to remediate hazardous inorganic/organic cross-contaminates in ASR.

Keywords: nano-sized metallic calcium, automobile shredder residue, organic/inorganic contaminants, immobilization, detoxification

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Authors: Shamima Akhter, Samira Bagheri, M. Shalauddin, Wan Jefrey Basirun

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In the present study, a nanocomposite of f-MWCNTs-Chitosan was prepared by the immobilization of Co(II) transition metal through self-assembly method and used for the simultaneous voltammetric determination of paracetamol (PA). The composite material was characterized by field emission scanning electron microscopy (FESEM) and energy dispersive X-Ray analysis (EDX). The electroactivity of cobalt immobilized f-MWCNTs with excellent adsorptive polymer chitosan was assessed during the electro-oxidation of paracetamol. The resulting GCE modified f-MWCNTs/CTS-Co showed electrocatalytic activity towards the oxidation of PA. The electrochemical performances were investigated using cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and differential pulse voltammetry (DPV) methods. Under favorable experimental conditions, differential pulse voltammetry showed a linear dynamic range for paracetamol solution in the range of 0.1 to 400µmol L⁻¹ with a detection limit of 0.01 µmol L⁻¹. The proposed sensor exhibited significant selectivity for the paracetamol detection. The proposed method was successfully applied for the determination of paracetamol in commercial tablets and human serum sample.

Keywords: nanomaterials, paracetamol, electrochemical technique, multi-wall carbon nanotube

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Authors: Kinga Mylkie, Pawel Nowak, Marta Z. Borowska

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The most important proteins in human serum responsible for drug binding are human serum albumin (HSA) and α1-acid glycoprotein (AGP). The AGP molecule is a glycoconjugate containing a single polypeptide chain composed of 183 amino acids (the core of the protein), and five glycan branched chains (sugar part) covalently linked by an N-glycosidic bond with aspartyl residues (Asp(N) -15, -38, -54, -75, - 85) of polypeptide chain. This protein plays an important role in binding alkaline drugs, a large group of drugs used in psychiatry, some acid drugs (e.g., coumarin anticoagulants), and neutral drugs (steroid hormones). The main goal of the research was to obtain magnetic nanoparticles coated with biopolymers in a chemically modified form, which will have highly reactive functional groups able to effectively immobilize the glycoprotein (acid α1-glycoprotein) without losing the ability to bind active substances. The first phase of the project involved the chemical modification of biopolymer starch. Modification of starch was carried out by methods of organic synthesis, leading to the preparation of a polymer enriched on its surface with aldehyde groups, which in the next step was coupled with 3-aminophenylboronic acid. Magnetite nanoparticles coated with starch were prepared by in situ co-precipitation and then oxidized with a 1 M sodium periodate solution to form a dialdehyde starch coating. Afterward, the reaction between the magnetite nanoparticles coated with dialdehyde starch and 3-aminophenylboronic acid was carried out. The obtained materials consist of a magnetite core surrounded by a layer of modified polymer, which contains on its surface dihydroxyboryl groups of boronic acids which are capable of binding glycoproteins. Magnetic nanoparticles obtained as carriers for plasma protein immobilization were fully characterized by ATR-FTIR, TEM, SEM, and DLS. The glycoprotein was immobilized on the obtained nanoparticles. The amount of mobilized protein was determined by the Bradford method.

Keywords: glycoproteins, immobilization, magnetic nanoparticles, polysaccharides

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Authors: Burcu Doymus, Fatma N. Kok, Sakip Onder

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Titanium and titanium-based materials are commonly used to replace or regenerate the injured or lost tissues because of accidents or illnesses. Hospital infections and strong bond formation at the implant-tissue interface are directly affecting the success of the implantation as weak bonding with the native tissue and hospital infections lead to revision surgery. The purpose of the presented study is to modify the surface of the titanium substrates with nano/microspheres for local drug delivery and to prevent hospital infections. Firstly, titanium surfaces were silanized with APTES (3-Triethoxysilylpropylamine) following the negatively charged oxide layer formation. Then characterization studies using Scanning Electron Microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR) were done on the modified surfaces. Secondly, microspheres/nanospheres were prepared with chitosan that is a natural polymer and having valuable properties such as non-toxicity, high biocompatibility, low allergen city and biodegradability for biomedical applications. Antibiotic (ciprofloxacin) loaded micro/nanospheres have been fabricated using emulsion cross-linking method and have been immobilized onto the titanium surfaces with different immobilization techniques such as covalent bond and entrapment. Optimization studies on size and drug loading capacities of micro/nanospheres were conducted before the immobilization process. Light microscopy and SEM were used to visualize and measure the size of the produced micro/nanospheres. Loaded and released drug amounts were determined by using UV- spectrophotometer at 278 nm. Finally, SEM analysis and drug release studies on the micro/nanospheres coated Ti surfaces were done. As a conclusion, it was shown that micro/nanospheres were immobilized onto the surfaces successfully and drug release from these surfaces was in a controlled manner. Moreover, the density of the micro/nanospheres after the drug release studies was higher on the surfaces where the entrapment technique was used for immobilization. Acknowledgement: This work is financially supported by The Scientific and Technological Research Council Of Turkey (Project # 217M220)

Keywords: chitosan, controlled drug release, nanosphere, nosocomial infections, titanium

Procedia PDF Downloads 106