Inhibition of biofilm, quorum-sensing, and swarming motility in pathogenic bacteria by magnetite, manganese ferrite, and nickel ferrite nanoparticles.

Resistance to antibiotics by pathogenic bacteria constitutes a health burden and nanoparticles (NPs) are being developed as alternative and multipurpose antimicrobial substances. Magnetite (Fe3O4 np), manganese ferrite (MnFe2O4 np) and nickel ferrite (NiFe3O4 np) NPs were synthesized and characterized using thermogravimetric analysis, transmission electron microscopy, Fourier transformed infra-red, and X-ray diffraction. The minimal inhibitory concentrations (MIC) ranged from 0.625 to 10 mg/mL against gram-positive (Staphylococcus aureus ATCC 25923 and Enterococcus faecalis ATCC 29212), gram-negative (Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853) and candida (Candida albicans ATCC 10239 and Candida tropicalis ATCC 13803) species. The NPs exhibited violacein inhibition against Chromobacterium violaceum CV12472 of 100% at MIC and reduced to 27.2% ± 0.8% for magnetite NPs, 12.7% ± 0.3% for manganese ferrite NPs and 43.1% ± 0.2% for nickel ferrite NPs at MIC/4. Quorum-sensing (QS) inhibition zones against C. violaceum CV026 were 12.5 ±0.6 mm for Fe3O4 np, 09.1 ± 0.5 mm for MnFe3O4 NP and 17.0 ± 1.2 mm for NiFe3O4 np. The NPs inhibited swarming motility against P. aeruginosa PA01 and biofilm against six pathogens and the gram-positive biofilms were more susceptible than the gram-negative ones. The NiFe2O4 np had highest antibiofilm activity against gram-positive and gram-negative bacteria as well as highest QS inhibition while Fe3O4 NP had highest biofilm inhibition against candida species. The synthesized magnetic NPs can be used in developing anti-virulence drugs which reduce pathogenicity of bacteria as well as resistance.

Fabrication of electrospun cellulose-derived nanofiber membranes with enhanced stability properties of arginase.

In this study, cellulose acetate (CA)/polyvinylpyrrolidone (PVP) and CA/PVP/Mn2+ nanofibers were produced by the electrospinning method, and these cellulose-derived membranes were used as carriers for arginase immobilization for the first time. The structural and morphological analysis of these cellulose-derived nanofibers were determined by attenuated total reflection-Fourier to transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), X-ray diffraction (XRD), and scanning electron microscopy (SEM). After the immobilization process, it was observed that the thermal properties of the cellulose-derived nanofibers especially improved. The optimum temperature value for free arginase was found to be 35 °C, and this value was found to be 40 °C for arginase immobilized cellulose-derived nanofibers. When the free arginase retained only about 35% of its activity at 50 °C and 60 °C after 60 min, arginase immobilized nanofibers protected 65% of their activity under the same conditions. In addition, arginase immobilized CA/PVP and CA/PVP/Mn2+ nanofibers managed to retain 50% of their activity even after 9 and 12 reuses, respectively.

Antibiofilm and anti-quorum sensing activities of polyethylene imine coated magnetite and nickel ferrite nanoparticles.

This study was aimed at synthesizing polyethyleneimine-coated magnetic nanoparticles and evaluating their effect on pathogenic bacteria. Polyethyleneimine-coated magnetite (PEIMnF) and nickel ferrite (PEINF) nanoparticles were succesfully synthesized and their surface groups, morphology and chemical structures were characterized using ATR-FTIR (Attenuated Total Reflectance Fourrier Transformed Infra-Red) and SEM (Scanning Electron Microscopy). TGA (Thermogravimetric analysis) was used to analyse the thermal behaviour and stability of synthesized nanomaterials. The minimal inhibitory concentration (MIC) values of the polyethylene imine coated magnetite and nickel ferrite nanomaterials against Staphylococcus aureus, Escherichia coli and Candida albicans was found to be 10 mg/mL. Both nanomaterials (PEIMnF and PEINF) showed very excellent and concentration-dependent biofilm inhibition especially at the highest test concentration of 10 mg/mL at which PEIMnF inhibited biofilm formation on E. coli (89.04 ± 0.50%), S. aureus (82.85 ± 2.42%) and C. albicans (91.37 ± 0.66%). At this concentration, PEINF equally inhibited biofilm formations of E. coli (90.48 ± 2.05%), S. aureus (87.04 ± 1.59%) and C. albicans (90.94 ± 1.03%). Only PEINF showed a concentration-dependent violacein inhibition with highest inhibition of 51.2 ± 3.5% at MIC and quorum sensing with inhibition zones of 16.3 ± 1.0 mm at MIC and 11.5 ± 0.5 mm at MIC/2 which could be attributed to the presence of nickel. The nanomaterials inhibited swimming and swarming motilities in Pseudomonas aeruginosa PA01 and it was found that at the same concentration, swimming inhibition was greater than swarming inhibitions and PEINF showed better inhibition than PEIMnF in both models. Polyethyleneimine-coated magnetite and nickel ferrite nanomaterials could be used in overcoming health problems associated with microbial infections and resistance.

Urease immobilized electrospun PVA/chitosan nanofibers with improved stability and reusability characteristics: an application for removal of urea from artificial blood serum.

Electrospun polyvinyl alcohol (PVA)/Chitosan nanofibers were successfully prepared and were used as carriers for the first time in urease immobilization. Also, urease immobilized electrospun PVA/Chitosan nanofibers were applied for the removal of urea from artificial blood serum by recycled reactor. The nanofibers were optimized and synthesized by electrospinning technique according to the operational parameters. The morphology and structure of the nanofibers were characterized by scanning electron microscopy (SEM), attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) and thermogravimetric analysis (TGA). Urease was immobilized on the nanofibers by adsorption and crosslinking methods. According to immobilization results, nanofiber enhanced urease stability properties like thermal stability, pH stability, and reusability. Urease immobilized electrospun PVA/Chitosan nanofiber protected its activity by 85% after 10 uses and 45% after 20 uses. Urea removal rates of artificial blood serum were as follows: 100% at 1st cycle, 95% at 2nd, 3rd and 4th cycles; 85% at the 5th cycle; 76% at the 6th cycle, and 65% at the last three cycles.

Synthesis and characterization of electrospun PVA/Zn2+ metal composite nanofibers for lipase immobilization with effective thermal, pH stabilities and reusability.

Polyvinyl alcohol (PVA)/Zn2+ electrospun nanofibers that were a kind of polymer/ionic metal composite was successfully embedded in the hybrid fibers for the first time in the literature, due to chemical interactions between PVA and Zn2+. Also, the nanofibers were used as carriers for the first time in enzyme immobilization. The nanofibers were optimized and synthesized by electrospinning technique according to the operational parameters like as PVA concentration (%), Zn2+ concentration (%), voltage (kV), needle tip-collector distance (cm) and injection speed (ml/h). The morphology and structure of the nanofibers were characterized by SEM, XRD, ATR-FTIR and TGA. Lipase was immobilized on the nanofibers by adsorption and crosslinking methods. According to immobilization results, nanofiber enhanced enzyme stability properties like as thermal stability, pH stability and reusability. Lipase immobilized nanofiber protected 90% of its activity after 14 reuses.

A comparative study for lipase immobilization onto alginate based composite electrospun nanofibers with effective and enhanced stability.

In this study, lipase was successfully immobilized on polyvinyl alcohol/alginate and polyethylene oxide/alginate nanofibers that were prepared by electrospinning. Results showed that nanofibers (especially polyvinyl alcohol/alginate) enhanced the stability properties of lipase. When the free lipase lost its all activity after 40-60min at high temperatures, both lipase immobilized nanofibers kept almost 65-70% activity at the same time. The lipase immobilized poly vinyl alcohol/alginate and polyethylene oxide/alginate nanofibers protected approximately all of their activities until pH 9. Lipase immobilized polyvinyl alcohol/alginate and polyethylene oxide/alginate nanofibers maintained 60% of their activities after 14 and 7 reuses, respectively. The morphology of nanofibers was characterized by Scanning Electron Microscope, Fourier Transform Infrared Spectroscopy and Thermal Gravimetric Analyzer. As a result, this nanofiber production method, electrospinning, is simple, versatile and economical for preparing appropriate carrier to immobilize the enzymes.

Synthesis and Characterisation of Biocompatible Polymer-Conjugated Magnetic Beads for Enhancement Stability of Urease.

We reported natural polymer-conjugated magnetic featured urease systems for removal of urea effectively. The optimum temperature (20-60 °C), optimum pH (3.0-10.0), kinetic parameters, thermal stability (4-70 °C), pH stability (4.0-9.0), operational stability (0-250 min), reusability (18 times) and storage stability (24 weeks) were studied for characterisation of the urease-encapsulated biocompatible polymer-conjugated magnetic beads. Also, the surface groups and chemical structure of the magnetic beads were determined by using attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR). The all urease-encapsulated magnetic beads protected their stability of 30-45 % relative activity at 70 °C. A significant increase was observed at their pH stability compared with the free urease for both acidic and alkaline medium. Besides this, their repeatability activity were approximately 100 % during 4(th) run. They showed residual activity of 50 % after 16 weeks. The importance of this work is enhancement stability of immobilised urease by biocompatible polymer-conjugated magnetic beads for the industrial application based on removal of urea.

A selective molecularly imprinted polymer for immobilization of acetylcholinesterase (AChE): an active enzyme targeted and efficient method.

In the present study, we immobilized acetylcholinesterase (AChE) enzyme onto acetylcholine removed imprinted polymer and acetylcholine containing polymer. First, the polymers were produced with acetylcholine, substrate of AChE, by dispersion polymerization. Then, the enzyme was immobilized onto the polymers by using two different methods: In the first method (method A), acetylcholine was removed from the polymer, and then AChE was immobilized onto this polymer (acetylcholine removed imprinted polymer). In the second method (method B), AChE was immobilized onto acetylcholine containing polymer by affinity. In method A, enzyme-specific species (binding sites) occurred by removing acetylcholine from the polymer. The immobilized AChE reached 240% relative specific activity comparison with free AChE because the active enzyme molecules bounded onto the polymer. Transmission electron microscopy results were taken before and after immobilization of AChE for the assessment of morphological structure of polymer. Also, the experiments, which include optimum temperature (25-65 °C), optimum pH (3-10), thermal stability (4-70 °C), kinetic parameters, operational stability and reusability, were performed to determine the characteristic of the immobilized AChE.

Synthesis and characterization of chitosan/TiO2 composite beads for improving stability of porcine pancreatic lipase.

The purpose of the present work is improving stability properties of porcine pancreatic lipase (triacylglycerol lipase, E.C.3.1.1.3) by immobilization on chitosan/TiO2 composite beads. The immobilization parameters were initial enzyme concentration (0.5-2 mg/ml), adsorption time (5-25 min), and glutaraldehyde concentration (1-4 % v/v). The optimum temperature (20-60 °C), optimum pH (3.0-10.0), kinetic parameters, thermal stability (4-70 °C), pH stability (4.0-9.0), and reusability (9 times) were investigated for characterization of immobilized lipase system. The optimum temperatures of free and immobilized lipase were 30 °C. The temperature profile of the immobilized lipase was spread over a large area. The optimum pH values for the free lipase and immobilized lipase were found to be 6.5 and 7.5, respectively. The thermal stability of immobilized lipase was evaluated, and it maintained 45 % activity at 70 °C. But, at this temperature, soluble lipase protected only 15 % activity. Also, the structural characterization of chitosan/TiO2 composite beads was analyzed with scanning electron microscope (SEM), X-ray diffraction (XRD), thermal gravimetric analysis (TGA), and attenuated total reflection Fourier transform infrared spectroscopy analysis (ATR-FTIR). The significance of this study is improving of stability properties of lipase for the industrial usage especially production of biodiesel and dairy products.

Improving of catalase stability properties by encapsulation in alginate/Fe3O4 magnetic composite beads for enzymatic removal of H2O2.

The aim of this study was enhancing of stability properties of catalase enzyme by encapsulation in alginate/nanomagnetic beads. Amounts of carrier (10-100 mg) and enzyme concentrations (0.25-1.5 mg/mL) were analyzed to optimize immobilization conditions. Also, the optimum temperature (25-50°C), optimum pH (3.0-8.0), kinetic parameters, thermal stability (20-70°C), pH stability (4.0-9.0) operational stability (0-390 min), and reusability were investigated for characterization of the immobilized catalase system. The optimum pH levels of both free and immobilized catalase were 7.0. At the thermal stability studies, the magnetic catalase beads protected 90% activity, while free catalase maintained only 10% activity at 70°C. The thermal profile of magnetic catalase beads was spread over a large area. Similarly, this system indicated the improving of the pH stability. The reusability, which is especially important for industrial applications, was also determined. Thus, the activity analysis was done 50 times in succession. Catalase encapsulated magnetic alginate beads protected 83% activity after 50 cycles.

TiO2 beads and TiO2-chitosan beads for urease immobilization.

The aim of the present study is to synthesize TiO2 beads for urease immobilization. Two different strategies were used to immobilize the urease on TiO2 beads. In the first method (A), urease enzyme was immobilized onto TiO2 beads by adsorption and then crosslinking. In the second method (B), TiO2 beads were coated with chitosan-urease mixture. To determine optimum conditions of immobilization, different parameters were investigated. The parameters of optimization were initial enzyme concentration (0.5; 1; 1.5; 2mg/ml), alginate concentration (1; 2; 3%), glutaraldehyde concentration (1; 2; 3% v/v) and chitosan concentration (2; 3; 4 mg/ml). The optimum enzyme concentrations were determined as 1.5mg/ml for A and 1.0mg/ml for B. The other optimum conditions were found 2.0% (w/v) for alginate concentration (both A and B); 3.0mg/ml for chitosan concentration (B) and 2.0% (v/v) for glutaraldehyde concentration (A). The optimum temperature (20-60°C), optimum pH (3.0-10.0), kinetic parameters, thermal stability (4-70°C), pH stability (4.0-9.0), operational stability (0-230 min) and reusability (20 times) were investigated for characterization. The optimum temperatures were 30°C (A), 40°C (B) and 35°C (soluble). The temperature profiles of the immobilized ureases were spread over a large area. The optimum pH values for the soluble urease and immobilized urease prepared by using methods (A) and (B) were found to be 7.5, 7.0, 7.0, respectively. The thermal stabilities of immobilized enzyme sets were studied and they maintained 50% activity at 65°C. However, at this temperature free urease protected only 15% activity.

Development of a new biosensor for determination of catalase activity.

Catalase is one of the major antioxidant enzymes that catalyzes the hydrolysis of H2O2. The aim of this study was to suggest a new method for the assay of catalase activity. For this purpose, an amperometric biosensor based on glucose oxidase for determination of catalase activity was developed. Immobilization of glucose oxidase was made by a cross-linking method with glutaraldehyde on a Clark-type electrode (dissolved oxygen probe). Optimization and characterization properties of the biosensor were studied and determination of catalase activity in defined conditions was investigated in artificial serum solution. The results were compared with a reference method.

Immobilization of bovine catalase onto magnetic nanoparticles.

The scope of this study is to achieve carrier-bound immobilization of catalase onto magnetic particles (Fe3O4 and Fe2O3NiO2 · H2O) to specify the optimum conditions of immobilization. Removal of H2O2 and the properties of immobilized sets were also investigated. To that end, adsorption and then cross-linking methods onto magnetic particles were performed. The optimum immobilization conditions were found for catalase: immobilization time (15 min for Fe3O4; 10 min for Fe2O3NiO2 · H2O), the initial enzyme concentration (1 mg/mL), amount of magnetic particles (25 mg), and glutaraldehyde concentration (3%). The activity reaction conditions (optimum temperature, optimum pH, pH stability, thermal stability, operational stability, and reusability) were characterized. Also kinetic parameters were calculated by Lineweaver-Burk plots. The optimum pH values were found to be 7.0, 7.0, and 8.0 for free enzyme, Fe3O4-immobilized catalases, and Fe2O3NiO2 · H2O-immobilized catalases, respectively. All immobilized catalase systems displayed the optimum temperature between 25 and 35°C. Reusability studies showed that Fe3O4-immobilized catalase can be used 11 times with 50% loss in original activity, while Fe2O3NiO2 · H2O-immobilized catalase lost 67% of activity after the same number of uses. Furthermore, immobilized catalase systems exhibited improved thermal and pH stability. The results transparently indicate that it is possible to have binding between enzyme and magnetic nanoparticles.

Sulfite determination by a biosensor based on bay leaf tissue homogenate: very simple and economical method.

Of all the food additives for which the FDA has received adverse reaction reports, the ones that most closely resemble true allergens are sulfur-based preservatives. Sulfites are used primarily as antioxidants to prevent or reduce discoloration of light-colored fruits and vegetables, such as dried apples and potatoes, and to inhibit the growth of microorganisms in fermented foods such as wine. This work aims to prepare an electrochemical biosensor based on bay leaf tissue homogenate that contains polyphenol oxidase enzyme abundantly for sulfite detection in foods. The principle of the biosensor is based on the inhibition effect of sulfites on polyphenol oxidase in the bioactive layer. Optimum conditions for the biosensor, such as temperature and pH, were investigated. Some stability parameters of the biosensor were also identified. The biosensor showed a linear calibration graph in the range of 25-100 microM sulfite. The biosensor presents a very simple, economical, reliable, and feasible method for sulfite detection in foods.

A biosensor based on Bay leaf (Laurus nobilis L.) tissue homogenate: improvement of the stability characteristics by a simple bio-imprinted technique.

Although enzymes are effective biocatalysts that are widely used in biosensors, a major drawback that hampers many of these biotechnological applications of enzymes is their limited stability. Applications that use very pure, high value proteins need to employ effective stabilization technology, primarily due to cost considerations and availability of the proteins used. For this purpose, interest in bio-imprinting techniques increases because it allows stability characteristics of enzymes to be improved. In this study, a bio-imprinted Bay leaf (Laurus nobilis L.) tissue homogenate biosensor was devised by a very simple way. For this purpose, the enzymes, polyphenol oxidases in the bay leaf tissue, were first complexed by using their competitive inhibitor, thiourea, in aqueous medium and then this enzyme was immobilized on gelatin by crosslinking with glutaraldehyde on a Clark-type oxygen electrode surface. Similarly, noncomplexed polyphenol oxidase with thiourea was also immobilized on a Clark-type oxygen electrode in the same conditions. The aim of the study was to prepare a new biosensor-based Bay leaf tissue homogenate and to improve the stability characteristics such as thermal stability, pH stability, and storage stability, of the biosensor by bio-imprinting method. The results showed that this simple technique should be effectively used to improve the stabilities of a biosensor.

A bio-imprinted urease biosensor: Improved thermal and operational stabilities.

Despite the increasing number of applications of biosensors in many fields, the construction of a steady biosensor remains still challenging. The high stability of molecularly bio-imprinted enzymes for its substrate can make them ideal alternatives as recognition elements for sensors. Urease (urea aminohydrolase, EC 3.5.1.5), which catalysis the hydrolysis of urea to ammonia and carbon dioxide, has been used in immobilized form in artificial kidney for blood detoxification. According to one report approximately half a million patients worldwide are being supported by haemodialysis. In this study, the enzyme of urease was first complexed by using a substrate analogue, thiourea, in aqueous medium and then this enzyme was immobilized on gelatin by crosslinking with glutaraldehyde on a glass electrode surface. Similarly, urease noncomplexed with thiourea was also immobilized on a glass electrode in the same conditions. The aim of the study was to compare the two biosensors in terms of their repeatability, pH stability and thermal stability, and also, linear ranges of two biosensors were compared with each other.

Two biosensors for phenolic compounds based on mushroom (Agaricus bisporus) homogenate: comparison in terms of some important parameters of the biosensors.

Interest in molecular imprinted polymer techniques has increased because they allows for the improvement of some stability characteristics of enzymes. The high stability of molecularly imprinted enzymes for a substrate can make them ideal alternatives as recognition elements for sensors. A bioimprinted mushroom tissue homogenate biosensor was constructed in a very simple way. For this purpose, sulfite was used. The enzyme, polyphenol oxidase, was first complexed by using a competitive inhibitor, sulfite, in aqueous medium and then the enzyme was immobilized on gelatin by crosslinking with glutaraldehyde on a glass electrode surface. Similarly, polyphenol oxidase uncomplexed with sulfite was also immobilized on a glass electrode in the same conditions. The aim of the study was to compare the two biosensors in terms of their repeatability and thermal, pH, and operational stability; also, the linear ranges of the two biosensors were compared with each other.

Immobilization of pancreatic lipase on chitin and chitosan.

In this study, porcine pancreatic lipase (EC 3.1.1.3) was immobilized on chitin and chitosan by adsorption and subsequent crosslinking with glutaraldehyde, which was added before (conjugation) or after (crosslinking) washing unbound proteins. Conjugation proved to be the better method for both supports. The properties of free and immobilized enzymes were also investigated and compared. The results showed that the pH optimum was shifted from 8.5 to 9.0 for both the immobilized enzymes. Also, the optimum temperature was shifted from 30 to 40 degrees C for chitin-enzyme and to 45 degrees C for chitosan-enzyme conjugates. The immobilization efficiency is low, but the immobilized enzymes have good reusability and stability (storage and operational). Besides these properties, the immobilized lipases were also suitable for catalyzing esterification reactions of fatty acids and fatty alcohols, both with a medium chain length. According to our results, esterification activities of immobilized lipases were two- and four-fold higher for chitosan- and chitin-enzyme, than for the free enzyme, respectively. The immobilization procedure shows a great potential for commercial applications of the immobilized lipase, a relatively low cost commercial enzyme.

Selective extraction of iron(III) from aqueous nitrate solution in the presence of cobalt(II), copper(II) and nickel(II) ions using bis(delta2-2-imidazolinyl)-5,5'-dioxime.

The feasibility of using bis(delta2-2-imidazolinyl)-5,5'-dioxime (H2L) for the selective extraction of iron(III) from aqueous solutions was investigated by employing an solvent-extraction technique. The extraction of iron(III) from an aqueous nitrate solution in the presence of metal ions, such as cobalt(II), copper(II) and nickel(II), was carried out using H2L in binary and multicomponent mixtures. Iron(III) extraction has been studied as a function of the pH, equilibrium time and extractant concentration. From the extracted complex species in the organic phase, iron(III) was stripped with 2 M HNO3, and later determined using atomic-absorption spectrometry. The extraction was found to significantly depend on the aqueous solution pH. The extraction of iron(III) with H2L increases with the pH value, reaching a maximum in the zone of pH 2.0, remaining constant between 2 and 3.5 and subsequently decreasing. The quantitative extraction of iron(III) with 5 x 10(-30 M H2L in toluene is observed at pH 2.0. H2L was found to react with iron(III) to form ligand complex having a composition of 1:2 (Fe:H2L).

Immobilization of phospholipase A2 on porous glass and its application for lowering serum cholesterol concentration.

Phospholipase A2 (PLA2; EC 3.1.1.4) is a lipolytic enzyme that hydrolysis the ester bond in sn-2 position of phospholipids. In this work, the PLA2 from hog pancreas was covalently coupled to porous glass. The properties of free and immobilized enzyme were also investigated and compared. The optimum pH and temperature were found as 8.5 and 50 degrees C, respectively for both free and immobilized enzyme. The immobilized enzyme had good properties that potential for medical application is considerable. Its use in lowering plasma cholesterol concentrations in blood samples was also demonstrated.