Biodegradation of Cibacron Blue 3GA by insolubilized laccase and identification of enzymatic byproduct using MALDI-ToF-MS: Toxicity assessment studies by Daphnia magna and Chlorella vulgaris.

The presented paper describes a detailed study on the use of immobilized laccase for effective degradation of Cibacron Blue 3GA dye. The amount of laccase loading on the cyclic carbonate groups containing poly(hydroxyethyl methacrylate-co-vinylene carbonate), p(HEMA-co-VC), microbeads was 27.8 mg g-1, and the retained immobilized enzyme activity was 73% compared to free enzyme. The toxicity of the dye and its byproducts were studied using Daphnia magna as test organism. The micro-algal growth inhibition was also studied using a green micro algae "Chlorella vulgaris". MALDI-ToF-MS was used to verify dye degradation byproducts. After 60 min of incubation period, Cibacron Blue 3GA (CB3GA) and its byproducts disappeared from the medium. After 60-min enzymatic treatment, the non-toxic nature of medium was confirmed by toxicity studies. On the other hand, the initial byproducts of the dye seemed to be more toxic than the later formed dye products. It should be noted that the information obtained from this study can be beneficial for understanding the initial degradation byproducts toxicities of the enzymatically treated dyes to provide information about environmental protection.

A facile and efficient method of enzyme immobilization on silica particles via Michael acceptor film coatings: immobilized catalase in a plug flow reactor.

A novel method was developed for facile immobilization of enzymes on silica surfaces. Herein, we describe a single-step strategy for generating of reactive double bonds capable of Michael addition on the surfaces of silica particles. This method was based on reactive thin film generation on the surfaces by heating of impregnated self-curable polymer, alpha-morpholine substituted poly(vinyl methyl ketone) p(VMK). The generated double bonds were demonstrated to be an efficient way for rapid incorporation of enzymes via Michael addition. Catalase was used as model enzyme in order to test the effect of immobilization methodology by the reactive film surface through Michael addition reaction. Finally, a plug flow type immobilized enzyme reactor was employed to estimate decomposition rate of hydrogen peroxide. The highly stable enzyme reactor could operate continuously for 120 h at 30 °C with only a loss of about 36 % of its initial activity.

Aminopyridine modified Spirulina platensis biomass for chromium(VI) adsorption in aqueous solution.

Chemical modification of Spirulina platensis biomass was realized by sequential treatment of algal surface with epichlorohydrin and aminopyridine. Adsorptive properties of Cr(VI) ions on native and aminopyridine modified algal biomass were investigated by varying pH, contact time, ionic strength, initial Cr(VI) concentration, and temperature. FTIR and analytical analysis indicated that carboxyl and amino groups were the major functional groups for Cr(VI) ions adsorption. The optimum adsorption was observed at pH 3.0 for native and modified algal biomasses. The adsorption capacity was found to be 79.6 and 158.7 mg g(-1), for native and modified algal biomasses, respectively. For continuous system studies, the experiments were conducted to study the effect of important design parameters such as flow rate and initial concentration of metal ions, and the maximum sorption capacity was observed at a flow rate of 50 mL h(-1), and Cr(VI) ions concentration 200 mg L(-1) with modified biomass. Experimental data fitted a pseudo-second-order equation. The regeneration performance was observed to be 89.6% and 94.3% for native and modified algal biomass, respectively.

Pathogen detection by core-shell type aptamer-magnetic preconcentration coupled to real-time PCR.

The presence of pathogenic bacteria is a major health risk factor in food samples and the commercial food supply chain is susceptible to bacterial contamination. Thus, rapid and sensitive identification methods are in demand for the food industry. Quantitative polymerase chain reaction (PCR) is one of the reliable specific methods with reasonably fast assay times. However, many constituents in food samples interfere with PCR, resulting in false results and thus hindering the usability of the method. Therefore, we aimed to develop an aptamer-based magnetic separation system as a sample preparation method for subsequent identification and quantification of the contaminant bacteria by real-time PCR. To achieve this goal, magnetic beads were prepared via suspension polymerization and grafted with glycidylmethacrylate (GMA) brushes that were modified into high quantities of amino groups. The magnetic beads were decorated with two different aptamer sequences binding specifically to Escherichia coli or Salmonella typhimurium. The results showed that even 1.0% milk inhibited PCR, but our magnetic affinity system capture of bacteria from 100% milk samples allowed accurate determination of bacterial contamination at less than 2.0 h with limit of detection around 100 CFU/mL for both bacteria in spiked-milk samples.

Activity and stability of urease entrapped in thermosensitive poly(N-isopropylacrylamide-co-poly(ethyleneglycol)-methacrylate) hydrogel.

Urease was entrapped in thermally responsive poly(N-isopropylacrylamide-co-poly(ethyleneglycol)-methacrylate), p[NIPAM-p(PEG)-MA], copolymer hydrogels. The copolymer membrane shows temperature-responsive properties similar to conventional p(NIPAM) hydrogels, which reversibly swell below and de-swell above the lower critical solution temperature of p(NIPAM) hydrogel at around 32 °C. The retained activities of the entrapped urease (in p[NIPAM-p(PEG)-MA]-4 hydrogels) were between 83 and 53% compared to that of the same quantity of free enzyme. Due to the thermo-responsive character of the hydrogel matrix, the maximum activity was achieved at around 25 °C with the immobilized urease. Optimum pH was the same for both free and entrapped enzyme. Operational, thermal and storage stabilities of the enzyme were found to increase with entrapment of urease in the thermoresponsive hydrogel matrixes. As for reusability, the immobilized urease retained 89% of its activity after ten repeated uses.

Immobilization of glucoamylase onto polyaniline-grafted magnetic hydrogel via adsorption and adsorption/cross-linking.

Glucoamylase (GA) was immobilized onto polyaniline (PANI)-grafted magnetic poly(2-hydroxyethylmethacrylate-co-glycidylmethacrylate) hydrogel (m-p(HEMA-GMA)-PANI) with two different methods (i.e., adsorption and adsorption/cross-linking). The immobilized enzyme preparations were used for the hydrolysis of "starch" dextrin. The amount of enzyme loading on the ferrogel was affected by the medium pH and the initial concentration of enzyme. The maximum loading capacity of the enzyme on the ferrogel was found to be 36.7 mg/g from 2.0 mg/mL enzyme solution at pH 4.0. The adsorbed GA demonstrated higher activity (59%) compared to adsorbed/cross-linked GA (43%). Finally, the immobilized GA preparations exhibited greater stability against heat at 55 °C and pH 4.5 compared to free enzyme (50 °C and pH 5.5), suggesting that the ferrogel was suitable support for immobilization of glucoamylase.

Design of a core-shell type immuno-magnetic separation system and multiplex PCR for rapid detection of pathogens from food samples.

We report an immuno-magnetic separation system developed by the immobilization of pathogen-specific antibodies on the core-shell magnetic beads. The magnetic beads were grafted with glycidylmethacrylate (GMA) using surface-initiated atom transfer radical polymerization (SI-ATRP). For immuno-magnetic separation (IMS) of target bacterial cells from others, antibodies for Escherichia coli and Salmonella enterica serovar Typhimurium cells were immobilized on the magnetic beads via glutaraldehyde coupling reaction. Our IMS system successfully separated Salmonella cells when the concentrations of target (i.e., Salmonella) and interfering (i.e., E. coli) cells were at the same level. Polymerase chain reaction (PCR) assays amplifying the rfb/rfbE region of the E. coli genome and a 647-bp fragment of the invA region of Salmonella were performed as the specific selection to accurately confirm the presence of E. coli and Salmonella, respectively. IMS and multiplex PCR methods can be used for specific and quantitative detection of pathogens from food samples. Thus, this study developed a reliable and direct system for rapid detection of Salmonella and E. coli in food samples. In addition, IMS method could be easily adapted to detect other pathogens by selecting the pertinent antibody.

Surface plasmon resonance aptasensor for Brucella detection in milk.

A Surface Plasmon Resonance (SPR) aptasensor was developed for the detection of Brucella melitensis (B. melitensis) in milk samples. Brucellosis is a bacterial zoonotic disease with global distribution caused mostly by contaminated milk or their products. Aptamers recognizing B. melitensis were selected following a whole bacteria-SELEX procedure. Two aptamers were chosen for high affinity and high specificity. The high affinity aptamer (B70 aptamer) was immobilized on the surface of magnetic silica core-shell nanoparticles for initial purification of the target bacteria cells from milk matrix. Another aptamer, highly specific for B. melitensis cells (B46 aptamer), was used to prepare SPR sensor chips for sensitive determination of Brucella in eluted samples from magnetic purification since direct injection of milk samples to SPR sensor chips is known for a high background unspecific signal. Thus, we integrated a quick and efficient magnetic isolation step for subsequent instant detection of B. melitensis contamination in one ml of milk sample by SPR with a LOD value as low as 27 ± 11 cells.

Reversible immobilization of uricase on conductive polyaniline brushes grafted on polyacrylonitrile film.

Polyacrylonitrile film (PAN) surfaces were modified with chemical polymerization of conductive polyaniline (PANI) in the presence of potassium dichromate as an oxidizing agent. The conductive films were used for immobilization of uricase. The surface resistance of the conductive film in this work was found to be 0.97 kΩ/cm. The maximum amount of immobilized enzyme on conductive film containing 2.4% PANI was about 216 µg/cm(2). The optimum pH for free and immobilized enzymes was observed at 7.0 and 7.5, respectively. The K (m) values for free and immobilized uricase were found to be 94 and 138 µM, respectively. V (max) values were calculated as 1.87 and 1.63 U/mg protein for the free and immobilized enzymes, respectively. Immobilized uricase exhibited ~68% of its original activity even after 2 months of storage at 4 °C while the free enzyme lost its initial activity within 4 weeks.

Poly(styrene-divinylbenzene) beads surface functionalized with di-block polymer grafting and multi-modal ligand attachment: performance of reversibly immobilized lipase in ester synthesis.

Fibrous poly(styrene-b-glycidylmethacrylate) brushes were grafted on poly(styrene-divinylbenzene) (P(S-DVB)) beads using surface-initiated atom transfer radical polymerization. Tetraethyldiethylenetriamine (TEDETA) ligand was incorporated on P(GMA) block. The ligand attached beads were used for reversible immobilization of lipase. The influences of pH, ionic strength, and initial lipase concentration on the immobilization capacities of the beads have been investigated. Lipase adsorption capacity of the beads was about 78.1 mg/g beads at pH 6.0. The Km value for immobilized lipase was about 2.1-fold higher than that of free enzyme. The thermal, and storage stability of the immobilized lipase also was increased compared to the native lipase. It was observed that the same support enzyme could be repeatedly used for immobilization of lipase after regeneration without significant loss in adsorption capacity or enzyme activity. A lipase from Mucor miehei immobilized on styrene-divinylbenzene copolymer was used to catalyze the direct esterification of butyl alcohol and butyric acid.

Fibrous polymer functionalized magnetic biocatalysts for improved performance.

Although enzymes are known for their excellent catalytic performance, industrial, medical or biotechnological applications should overcome some drawbacks like long-term stability under specific conditions of the application. Immobilized enzymes have offered advantages over soluble counterparts in many industrial and laboratory scale applications by increasing operational stability and reusability. When the immobilization matrix has magnetic properties, an additional advantage is obtained as simpler processing. Iron-based superparamagnetic nano-sized particles has large surface area for bio-compatible applications are especially in focus. Adding nanofibrous polymers to magnetic nanoparticles has been an excellent way to increase efficiency of biocatalyst immobilization by further increasing loading capacity. This chapter explains various magnetic enzyme-nanoparticles based preparations with potential for future industrial applications like invertase, lipase and as model studies and focus on the nanofibrous polymer brush grafting as a way to increase catalytic efficiency of magnetic nanoparticles.

Catalytic Activity of Immobilized Chymotrypsin on Hybrid Silica-Magnetic Biocompatible Particles and Its Application in Peptide Synthesis.

In this work, novel silica hybrid magnetic particles with biocompatible surface were designed as a support for enzyme immobilization, and the immobilized chymotrypsin (CT) performance was clarified as a model biocatalyst. CT is used in food technology for drink clarification and protein hydrolysis. The enzyme was directly immobilized onto polydopamine-grafted magnetic silica particles (MNP@SiO2@PDA-CT) via the Schiff base reaction. Immobilized enzyme had broadened for both pH and temperature profiles compared with the native CT. The MNP@SiO2@PDA-CT system also improved in thermostability compared with the native enzyme. The immobilized CT was operated in a continuous enzyme reactor (CER) for the hydrolysis of different proteins (i.e., cytochrome c (Cyt c), human serum albumin (HSA), human immunoglobulin G (HIgG), and lysozyme (Lys)). The peptide synthesis rate was shown to decrease as a function of increasing flow rate. The catalytic activity of the CER remained stable for 6.0 h in a continuous operation period; thus, the presented method may increase applicability in the food technology area. The immobilized CT in the CER showed a good hydrolysis performance for all the tested model proteins. The peptides hydrolyzed from the tested proteins were analyzed with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF-MS). Results show that the MNP@SiO2@PDA-CT system also permits for the applicability in the area of proteomic research.

Hydrophilic spacer-arm containing magnetic nanoparticles for immobilization of proteinase K: Employment for speciation of proteins for mass spectrometry-based analysis.

Proteinase K (ProK) is used for the degradation of proteins in cell lysates to isolate nucleic acids, and for the speciation of proteins for mass spectrometry analysis. In this work, a novel and sensitive immobilization process was developed for examination of protein mixtures by combining MALDI-ToF-MS and nLC-TIMS-ToF-MS/MS systems. To achieve these goals, magnetic nanoparticles (MPs) were prepared via thermal coprecipitation reaction under alkaline condition. The MPs were grafted with a silica layer (i.e., 3-(2,3-epoxypropoxy) propyltrimethoxysilane; EPTES) containing reactive epoxy groups. Then, the silica-grafted magnetic particles were coated with a long chain hydrophilic poly(ethylene glycol) diamine polymer (PEGDAP). The prepared materials were characterized by the Brunauer-Emmett-Teller (BET) method, X-ray diffraction (XRD), scanning electron microscopy (SEM) and attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy. The VSM data show that the MPs@EPTES@PEGDAP has paramagnetic performance with a saturation magnetization of approximately 32.3 emu g-1. Proteinase K (EC 3.4.21.64) was covalently immobilized on the MPs@EPTES by reaction of its epoxy groups with amine groups of the enzyme. On the other hand, the ProK was immobilized on the MPs@EPTES@PEGDAP after activation with glutaraldehyde and the immobilization reaction was realized by the coupling reaction between aldehyde groups of the support and amine groups of the enzyme. The amounts of immobilized ProK on the MPs@EPTES and MPs@EPTES@PEGDAP were found to be 27.4 and 19.6 mg g-1and the retained activities were determined to be 29 and 87%, respectively. For the first time, some important features such as thermal and storage stabilities, reusability and potential use in protein speciation for mass spectrometry-based techniques were also evaluated. For examples, after six weeks of storage at 4 °C, the immobilized ProK on the MPs@EPTES@PEGDAP-ProK still maintained 59% of its initial activity. However, at the end of the six-week storage period, its free counterpart had lost all of its initial activity. The immobilized ProK was also utilized for degradation and identification of model proteins (i.e., α-2-HS glycoprotein, ß-casein, bovine serum albumin and immunoglobulin). After enzymatic treatment, the digested peptides were analyzed and mapped by using nLC-TIMS-ToF-MS/MS systems.

Rapid and label-free detection of Brucella melitensis in milk and milk products using an aptasensor.

In this work, a novel quartz crystal microbalance (QCM) aptasensor is designed for the diagnosis of Brucella melitensis bacteria, which affects the Mediterranean fever (brucellosis) from the zoonotic diseases that are very common in the Middle East Countries. The method is based on the selection of B. melitensis bacterium from solutions using B. melitensis specific binding aptamer (Apt) attached magnetic nanoparticles. The surface of the magnetic nanoparticles (i.e.,Fe3O4) was modified by 3-aminopropyltriethoxysilane (APTES) and then grafted with a hydrophilic macromonomer poly(ethyleneglycol)-methacrylate (PEG-MA) as a first block polymer and glycidylmethacrylate (GMA) as a second block functional polymer via atom transfer radical polymerization (ATRP) method [Fe3O4 @SiO2 @p(PEG-MA-GMA)], then, the specific binding aptamer was immobilized. The aptamer immobilized magnetic nanoparticles were used for the pre-concentration of the target bacterium, and the same aptamer sequence was also immobilized on the QCM chip and used for the quantitative detection of B. melitensis using QCM aptasensor. The detection limits of the QCM aptasensor were in the range 1.02-1.07 CFU mL-1, with recoveries up to 79%. The synthesized [Fe3O4 @SiO2 @p(PEGMA-GMA)] nanoparticles showed a good permanence and high isolation recoveries for the pull down of the target bacterium from food samples, after recycling eight times. The method was successfully applied to target bacterium determinations in milk and milk product samples.

Design of an aptamer-based magnetic adsorbent and biosensor systems for selective and sensitive separation and detection of thrombin.

An aptasensor was designed for sensitive detection of thrombin using in biological fluids by integrating a magnetic aptamer-microbeads. To achieve this goal, the surface of gold plated QCM crystals was coated with L-cysteine and a thrombin binding DNA aptamer was immobilized on the L-cysteine coated QCM crystals surface via glutaraldehyde coupling. The binding interactions of thrombin to QCM crystals were characterized. Magnetic poly(2-hydroxyethyl methacrylate-ethylene glycol dimethacrylate-vinylene carbonate), Mp(HEMA-EGDMA-VC) microbeads were synthesized and thrombin binding aptamer (TBA) was immobilized. The Mp(HEMA-EGDMA-VC)-TBA microbeads were effectively adsorbed thrombin from serum in a relatively short contact time (ca. 5.0 min), and the eluted protein from Mp(HEMA-EGDMA-VC)-TBA was transferred to the QCM aptasensor that showed a specific detection of thrombin from serum. The detection limit of thrombin using aptasensor was 1.00 nmol L-1. The calculation dissociation constant of the aptasensor was 68.5 nmol L-1. The selectivity of the aptasensor system was tested with three different proteins (i.e., elastin, immunoglobulin G (IgG) and human serum albumin (HSA)) and showed high specificity to thrombin. The aptasensor was regenerated by washing with NaOH solution, and repeatedly used until 20 cycles without a change in the performance.

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

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 degrees 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.

Fast and Sensitive Detection of Salmonella in Milk Samples Using Aptamer-Functionalized Magnetic Silica Solid Phase and MCM-41-Aptamer Gate System.

General detection methods for S. enterica include PCR analysis, immunologic methods, solid culturing techniques, and various microscopic studies. Milk and other food samples demonstrate an especially difficult challenge for direct detection, resulting from high biological contents. In this report, we aimed for fast detection of pathogen cells through an efficient magnetic capture and subsequent quick detection based on aptamer affinity. The Fe3O4@SiO2@pGMA and MCM-41 particles were prepared separately and used for preconcentration and detection, respectively. Aptamer oligonucleotide sequences against S. enterica were fixed on both amine-functionalized MCM-41 and Fe3O4@SiO2@pGMA particles via glutaraldehyde coupling. The captured Salmonella cells were determined by a fluorescent homogeneous assay in the samples by aptamer-gated MCM-41 silica particles. Our method achieved a sensitive assay to detect Salmonella cells in milk samples as low as 103 CFU/ml without any culturing. Hence, the proposed sensing strategy might be an efficient platform for pathogen detection in a food matrix.

Biosorption of Reactive Red-120 dye from aqueous solution by native and modified fungus biomass preparations of Lentinus sajor-caju.

The capacities and mechanisms of native and treated white-rot fungus "Lentinus sajur-caju" biomass preparations in removing of textile dye (i.e. Reactive Red-120) from aqueous solution was investigated with different parameters, such as adsorbent dosage, pH, temperature and ionic strength. In the batch system, the maximum dye uptake on all the tested fungal biomass preparations was observed at pH 3.0, and the dye uptake capacities of the biosorbents (at 800 mg/l dye concentration) were found to be 117.8, 182.9, 138.6 and 57.2mg/g for native and heat-, acid- and base-treated dry fungal preparations, respectively. The uptake capacities order of the fungal preparations for the dye were found as heat-treated>acid-treated>native>base-treated. The Langmuir, Freundlih and Temkin adsorption models were used for the mathematical description of the biosorption equilibrium. The Freundlich and Temkin models were able to describe the biosorption equilibrium of Reactive Red-120 on the fungal biomass preparations. The dye biosorption on the fungal biomass preparations followed second-order kinetic model and equation.

Kinetics of mercury ions removal from synthetic aqueous solutions using by novel magnetic p(GMA-MMA-EGDMA) beads.

Poly(glycidylmethacrylate-methylmethacrylate), p(GMA-MMA-EGDMA), magnetic beads were prepared via suspension polymerization in the presence of ferric ions. The epoxy groups of the beads were converted into amino groups via ring opening reaction of the ammonia and, the aminated magnetic beads were used for the removal of Hg(II) ions from aqueous solution in a batch experiment and in a magnetically stabilized fluidized bed reactor (MFB). The magnetic p(GMA-MMA-EGDMA) beads were characterized with scanning electron microscope (SEM), FT-IR and ESR spectrophotometers. The optimum removal of Hg(II) ions was observed at pH 5.5. The maximum adsorption capacity of Hg(II) ions by using the magnetic beads was 124.8+/-2.1 mgg(-1) beads. In the continuous MFB reactor, Hg(II) ions adsorption capacity of the magnetic beads decreased with an increase in the flow-rate. The maximum adsorption capacity of the magnetic beads in the MFB reactor was 139.4+/-1.4 mgg(-1). The results indicate that the magnetic beads are promising for use in MFB for removal of Hg(II) ions from aqueous solution and/or waste water treatment.