Electrospun microfibrous poly(styrene-alt-maleic anhydride)/poly(styrene-co-maleic anhydride) mats tailored for enzymatic remediation of waters polluted by endocrine disruptors

Novel bicomponent microfibrous mats containing targeted amount of reactive maleic anhydride groups were prepared by electrospinning of mixed solutions of poly(styrene-alt-maleic anhydride) and poly(styrene-co-maleic anhydride). Then, amino-functionalized P(St-alt-MA)/P(St-co-MA) mats were obtained by reaction with p-phenylenediamine. ATR-FTIR and XPS spectroscopy were used to characterize pristine and modified P(St-alt-MA)/P(St-co-MA) mats. On the next step, laccase from Trametes versicolor was covalently attached onto the modified mats; the average amount of immobilized enzyme was 40 ± 0.7 mg/g mat. The catalytic activity of the immobilized enzyme was studied in respect to bisphenol A (BPA) endocrine disruptor. The optimum activity of the immobilized enzyme was reached at maximum flow rate of 1.3 mL/s. After 90 min the BPA concentration was reduced by 60% and the catalytic activity of microfibrous mats remained stable for about 30 successive reuses. In addition, the relative activity of laccase immobilized on the microfibrous mats was displayed in a broader pH range as compared to that of the free one.     

Electrospun Mats from Styrene/Maleic Anhydride Copolymers: Modification with Amines and Assessment of Antimicrobial Activity

New antimicrobial microfibrous electrospun mats from styrene/maleic anhydride copolymers were prepared. Two approaches were applied: (i) grafting of poly(propylene glycol) monoamine (Jeffamine® M‐600) on the mats followed by formation of complex with iodine; (ii) modification of the mats with amines of 8‐hydroxyquinoline or biguanide type with antimicrobial activity. Microbiological screening against S. aureus, E. coli and C. albicans revealed that both the formation of complex with iodine and the covalent attachment of 5‐amino‐8‐hydroxyquinoline or of chlorhexidine impart high antimicrobial activity to the mats. In addition, S. aureus bacteria did not adhere to modified mats.

     

Immobilization of bromelain onto porous copoly(γ-methyl-l-glutamate/l-leucine) beads

Water-insoluble bromelain was prepared by immobilizing bromelain onto the surface of porous copoly(γ-methyl-l-glutamate/l-leucine) (ML) beads with and without spacer. The mode of the immobilization between bromelain and porous copolypeptide ML beads was covalent fixation. The relative activity and the stability of the immobilized bromelain was investigated. The retained activity of the bromelain covalently immobilized by the azide method was found to be excellent toward a small ester substrate, N-benzyl-l-arginine ethyl ester, but rather low toward casein, a high molecular weight substrate. The values of the Michaelis constant K_m and the maximum reaction velocity V_m for free and immobilized bromelain on the porous copolypeptide ML beads were estimated. Apparent K_m was larger for immobilized bromelain than for the free one, while V_m was smaller for the immobilized bromelain. The thermal stability of the covalently immobilized bromelain was higher than that of the free bromelain. The initial enzymatic activity of the immobilized bromelain remained approximately unchanged with storage time, when the batch enzyme reaction was performed repeatedly, indicating the excellent durability.     

Immobilization of the enzyme L-asparaginase fromE. coli on polysaccharides IV. Covalent binding with dialdehyde-dextran

The synthesis has been effected of immobilizedE. coli L-asparaginase on medical dextran — poliglyukin. The influence of the bound polymer on some physicochemical properties of the final products have been studied. An increased resistance to heat and stability on storage of the immobilized forms of L-asparagine in comparison with a native enzyme has been found. It has been shown that the polymer modification of L-asparaginase leads to a decrease in the antigenic affinity of the immobilized enzyme as compared with the native enzyme.     

Effect of a spacer on phthalocyanine functionalized cellulose nanofiber mats for decolorizing reactive dye wastewater

We report a novel cobalt tetraaminophthalocyanine (CoPc) functionalized nanomaterial by spacer-arm immobilization of CoPc onto cellulose nanofiber mats. The spacer-arm was attached through the reaction of tetraethylenepentamine with oxidized cellulose nanofiber mats. CoPc was then covalently immobilized onto the spacer-arm using glutaraldehyde. The functionalization processes on the nanofiber mats were monitored by attenuated total reflection Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy. This CoPc functionalized nanomaterial (CoPc-spacer-NM) was used for decoloration of reactive dye wastewater. Incorporation of the spacer-arm resulted in enhanced decoloration with respect to directly immobilized CoPc onto the cellulose nanofiber mats (CoPc-NM). Compared with CoPc-NM, CoPc-spacer-NM shows much higher adsorption capacity when conducted under acidic conditions, which enhances the catalytic oxidation rate of reactive dye when H₂O₂ was used as an oxidant. Reactive dye wastewater can also be efficiently decolorized by the CoPc-spacer-NM/H₂O₂ system under basic conditions, despite a relatively weak adsorption capacity. Electron paramagnetic resonance results suggested that the catalytic oxidation process involves the formation and reaction of hydroxyl radicals. Gas chromatography–mass spectrometry showed the main products of the catalytic oxidation of reactive red X-3B were biodegradable aliphatic acids, such as oxalic acid, malonic acid and maleic acid.     

Immobilization of cellulase using acrylamide grafted acrylonitrile copolymer membranes

Immobilization of cellulase onto acrylamide grafted acrylonitrile copolymer (PAN) membranes by means of glutaraldehyde has been studied. The bound cellulase was verified by X-ray photoelectron spectroscopy. The activities of free cellulase and immobilized cellulase are determined by measuring the amount of glucose made from carboxymethyl cellulase in the given conditions. Results show that immobilization conditions had some effects on the activity of immobilized cellulase. The immobilized cellulase had a higher K_m than free cellulase (0.02 mg/ml) did. The immobilized cellulase had better stability with respect to pH or temperature than free cellulase.     

Man-tailored cellulose-based carriers for invertase immobilization

Cellulose-based carriers Granocel were specially prepared and optimised for covalent immobilization of enzymes. The effects of carrier characteristics such as pore size, chemistry of anchor groups and their density on invertase immobilization efficiency were evaluated. It was found that the preferential adsorption/binding of the enzyme to a carrier during coupling and its activity after immobilization depended on microenvironmental effects created by hydrophilic surface of the carrier, functional groups and their activators. The best preparations (activity approx. 300 U/mL, high storage stability) were obtained for NH₂-Granocel activated with glutaraldehyde. It is probably due to Granocel modification with pentaethylenehexamine that gave a 19-atom spacer arm. The enzyme concentration in coupling mixture was optimised as well. The kinetic parameters of sucrose hydrolysis for native and immobilized invertase were evaluated. Compared to the native invertase, K _m value of immobilized enzyme was only twice higher with about three times lower substrate inhibition. Reaction runs in a well mixed batch reactors with native and immobilized invertase showed slightly slower reaction rate in the case of the enzyme covalently bound to Granocel. Very good stability of cellulose-based carrier was proved experimentally by 20 successive reaction runs in a batch reactor.     

Spectroscopic quantification of covalently immobilized oligonucleotides

Quantitative determination of surface coverage, film thickness and molecular orientation of DNA oligomers covalently attached to aminosilane self‐assembled monolayers has been obtained using complementary infrared and photoelectron studies. Spectral variations between surface immobilized oligomers of the different nucleic acids are reported for the first time. Carbodiimide condensation was used for covalent attachment of phosphorylated oligonucleotides to silanized aluminum substrates. Fourier transform infrared (FTIR) spectroscopy and x‐ray photoelectron spectroscopy (XPS) were used to characterize the surfaces after each modification step. Infrared reflection–absorption spectroscopy of covalently bound DNA provides orientational information. Surface density and layer thickness are extracted from XPS data. The surface density of immobilized DNA, 2–3 (×10¹³) molecules cm^?2, was found to depend on base composition. Comparison of antisymmetric to symmetric phosphate stretching band intensities in reflection–absorption spectra of immobilized DNA and transmission FTIR spectra of DNA in KBr pellet indicates that the sugar–phosphate backbone is predominantly oriented with the sugar–phosphate backbone lying parallel to the surface, in agreement with the 10–20 Å DNA film thickness derived from XPS intensities. Copyright © 2004 John Wiley & Sons, Ltd.     

An optical biosensor for dichlovos using stacked sol-gel films containing acetylcholinesterase and a lipophilic chromoionophore

An optical biosensor consisting of a chromoionophore (ETH5294) (CM) doped sol-gel film interfaced with another sol-gel film immobilized with acetylcholinesterase (AChE) was employed to detect the insecticide dichlorvos. The main advantage of this optical biosensor is the use of a sol-gel layer with immobilized CM that possesses lipophilic property. The highly lipophilic nature of the CM and its compatibility with the sol-gel matrix has prevented leaching, which is frequently a problem in optical sensor construction based on pH indicator dyes. The immobilization of the indicator and enzyme was simple and need no chemical modification. The CM layer is pH sensitive and detects the pH changes of the acetylcholine chloride (AChCl) substrate when hydrolyzed by AChE layer deposited above. In the absence of the AChE layer, the pH response of the CM layer is linear from pH 6 to 8 (R² = 0.98, n = 3) and it showed no leaching of the lipophilic chromoionophore. When the AChE layer is deposited on top, the optical biosensor responds to AChCl with a linear dynamic range of 40-90 mM AChCl (R² = 0.984, n = 6). The response time of the biosensor is 12 min. Based on the optimum incubation time of 15 min, a linear calibration curve of dichlorvos against the percentage inhibition of AChE was obtained from 0.5 to 7 mg/L of dichlorvos (17-85% inhibition, R² = 0.991, n = 9). The detection limit for dichlorvos was 0.5 mg/L. The results of the analysis of 1.7-6.0 mg/L of dichlorvos using this optical biosensor agreed well with a gas chromatography-mass spectrometry detection method.     

基于固定化酶的乙酰胆碱酯酶抑制剂体外筛选模型的建立

以可重复使用的固定化酶代替游离态酶, 建立一种基于比色分析的乙酰胆碱酯酶(AChE)抑制剂体外筛选新模型. 采用以氨基化硅胶为载体固定的AChE优化了实验条件, 用AChE抑制剂阳性对照物他克林和毒扁豆碱对该模型进行验证, 还对模型技术参数进行评价, 并将新模型用于单体化合物及天然产物粗提物AChE抑制活性评价. 结果表明, 最佳实验条件为: 固定化酶用量55 μL, 底物浓度5 mmol/L, 甲醇、 乙醇及体积分数不高于6%的二甲基亚砜水溶液均可作为样品溶剂; 模型验证及模型技术参数评价结果良好, 该模型对AChE抑制剂筛选有较好的特异性和灵敏度, 可用于筛选AChE抑制剂. 该模型具有适用性强、 固定化酶可重复使用及结果可靠等优点, 是单体化合物及天然产物粗提物AChE抑制剂活性评价的有效方法.     

Cobalt(II) complexation with immobilized proteinases of Candida albicans

Complexation of cobalt(II) ion with proteolytic enzymes of Candida albicans (SAP C.alb.) of induced (ISAP C.alb.) and constitutive (CSAP C.alb.) types immobilized on the cellulose nitrate membrane surface has been studied. The maximal sorption capacity of cellulose nitrate membranes with covalently immobilized induced proteinase ISAP C.alb. with respect to Co(II) ions is 16.5 ??mol/cm², and that for CSAP C.alb. is 27.7 ??mol/cm². The model of fixed polydentate centers has been used for describing complex formation. The complexation constants (?? _n ) and the average number of immobilized ligands coordinated to one metal atom (n) have been determined. The specificity of binding of immobilized enzyme molecules to Co(II) ion has been assessed.     

trans,trans-2,4-Hexadiene incorporation on enzymes for site-specific immobilization and fluorescent labeling

Lipase B from Candida antarctica (CAL-B) has been site-directedly modified by the introduction of a trans,trans-hexadiene moiety onto lipase molecules, identified by MALDI-TOF. This modification on CAL-B permitted its immobilization on Q-Sepharose supports in excellent yields (>95%) when native lipase was not immobilized at pH 7 and 25 °C. After the entire modification procedure, the catalytic activity of the protein on the solid support was surprisingly increased 2-fold. A tailor-made maleimide-fluorophore derivative was specifically covalently linked to the protein in high yield via a selective Diels-Alder reaction in aqueous media. Furthermore, the NBD-labeled-CAL-B was also immobilized on the ionic support, retaining around 80% of the specific activity. The preparation of this labeled-CAL-B was also possible by a Diels-Alder reaction on solid phase in excellent yields.     

Affinity Covalent Immobilization of Glucoamylase onto ρ-Benzoquinone-Activated Alginate Beads: II. Enzyme Immobilization and Characterization

A novel affinity covalent immobilization technique of glucoamylase enzyme onto ρ-benzoquinone-activated alginate beads was presented and compared with traditional entrapment one. Factors affecting the immobilization process such as enzyme concentration, alginate concentration, calcium chloride concentration, cross-linking time, and temperature were studied. No shift in the optimum temperature and pH of immobilized enzymes was observed. In addition, K _m values of free and entrapped glucoamylase were found to be almost identical, while the covalently immobilized enzyme shows the lowest affinity for substrate. In accordance, V _m value of covalently immobilized enzyme was found lowest among free and immobilized counter parts. On the other hand, the retained activity of covalently immobilized glucoamylase has been improved and was found higher than that of entrapped one. Finally, the industrial applicability of covalently immobilized glucoamylase has been investigated through monitoring both shelf and operational stability characters. The covalently immobilized enzyme kept its activity over 36 days of shelf storage and after 30 repeated use runs. Drying the catalytic beads greatly reduced its activity in the beginning but recovered its lost part during use. In general, the newly developed affinity covalent immobilization technique of glucoamylase onto ρ-benzoquinone-activated alginate carrier is simple yet effective and could be used for the immobilization of some other enzymes especially amylases.     

Enzyme inhibitor screening by electrospray mass spectrometry with immobilized enzyme on magnetic silica microspheres

In this study, a novel technique for screening inhibitors by electrospray mass spectrometry (ESI-MS) with immobilized enzyme on magnetic microspheres has been demonstrated. First, the model enzyme acetylcholinesterase (AChE) is immobilized onto the 3-glycidoxypropyltrimethoxysilane (GLYMO)-modified magnetic silica microspheres. AChE activity was monitored by biochemical assay that is based on mixing of AChE immobilized microspheres and model substrate acetylcholine, separating and detecting the product through ESI-MS. Stability of the enzyme-immobilized microspheres was investigated. No apparent loss of enzyme activity was observed after fivefold reuse of AChE-immobilized microspheres. The enzyme-immobilized bioassay was used to effectively identify AChE inhibitors among two standard samples, huperzine A and huperzine B, and their source herbal Huperzia serrata, all of which were spiked into the substrate. The inhibition was determined by measuring a decrease of product formation using ESI-MS.     

Antimicrobial Activity of Electrospun Poly(butylenes succinate) Fiber Mats Containing PVP-Capped Silver Nanoparticles

In this study, biodegradable poly(butylenes succinate) (PBS) fiber mats containing silver nanoparticles (AgNPs) were prepared by the electrospinning process. Small AgNPs (<10 nm) were simply synthesized using polyvinylpyrrolidone as the capping agent as well as the reductant. The morphology of the PBS-AgNPs fiber mats and the distribution of the AgNPs were well characterized by TEM and SEM. The release of Ag from the PBS fiber mats was quantitively determined by ICP. The PBS fiber mats with 0.29 % AgNPs content showed strong antimicrobial activity against both gram-positive Staphylococcus aureus and gram-negative Escherichia coli with the efficacy as high as 99 %. The effective bactericidal activity on E. coli was demonstrated for a short contacting time with the PBS-AgNPs fiber mats. In addition, the long-term release performance of Ag from the fiber mats can keep inhibiting the bacterial growth in the mats over a long period of time.     

Synthesis of an immobilized Bombyx mori pheromone-binding protein liquid chromatography stationary phase

The pheromone-binding protein from the silkworm moth, Bombyx mori (BmorPBP) has been covalently bonded to a liquid chromatographic stationary phase. The resulting column was evaluated using radiolabeled bombykol and the immobilized protein retained its ability to bind this ligand. The data also demonstrate that the BmorPBP column was able to distinguish between four compounds, and rank them in their relative order of affinity for the protein from highest to lowest: bombykol > bombykal > 1-hexadecanol > (Z,E)-5,7-dodecadien-1-ol, and that the immobilized BmorPBP retained its pH-dependent conformational mobility.The results of this study demonstrate that pheromone-binding protein from the silkworm moth, Bombyx mori and an odorant binding protein (OBP) obtained from the female mosquito Culex quinquefasciatoes have been immobilized on a silica support with retention of ligand-binding activity. The data indicate that proteins from non-mammalian organisms can be used to create liquid chromatography affinity columns.     

Chemical modification of acetylcholinesterase with methoxypolyethylene glycol

Acetylcholinesterase (AChE) (EC 3.1.1.7) was modified with activated monomethoxypolyethylene glycol (mPEG). A decrease of 50% in the catalytic activity was measured during the coupling reaction and the change in the surface properties of AChE was used to separate by hydrophobic interaction chromatography the native and the modified enzyme. The native and the modified enzymes were found to have the same optimalcatalytic conditions. Moreover, the Michaelis constant of both enzymes were similar, whereas theV _m and the bimolecular-velocity constant calculated for organophosphorus inhibitors were slightly higher for the modified AChE. Finally, the modification with mPEG did not improve the thermal stability, whereas the stability in a few organic solvents increased.     

Preparation and characterization of lipase immobilized on reversibly soluble-insoluble N-(2-carboxylbenzoyl) chitosan

N-(2-carboxylbenzoyl) chitosan (CBC), a reversibly soluble-insoluble polymer with pH change, was prepared by modifying chitosan backbone with phthalic anhydride and employed as carrier for lipase immobilization. The obtained CBC exhibited reversible solubility in aqueous solution; it was soluble at pH above 3.8 and precipitated at pH below 3.4. The porcine pancreatic lipase was covalently immobilized on CBC with glutaraldehyde as the crosslinking agent. Under the optimal immobilization condition, the retention activity of the immobilized lipase was found to be 69.8 %. The maximum activity of lipase immobilized on CBC was observed at 40 °C, pH 8.0, while the free lipase presented maximum activity at 37 °C, pH 7.5. The lipase immobilized on CBC exhibited improved thermal and storage stabilities and retained 58.7 % of its initial activity after 9 times of repeated use.     

Spectrophotomeric Assay of Aflatoxin B1 Using Acetylcholinesterase Immobilized on Standard Microplates

Aflatoxins are a group of hepatotoxic secondary metabolites produced by molds Aspergillus. During inappropriate storage or processing of food and beverages, aflatoxins can be distributed and become a serious health problem. Disparate analytical techniques were prepared for the assay in the past. Here, a method based on inhibition of enzyme acetylcholinesterase (AChE) is performed allowing fast and simple prove of aflatoxins presence. For the assay purposes, AChE was immobilized on standard polystyrene microplates using gelatin as a stabilizing substance. Both free and immobilized AChEs were used and compared one to each other. Immobilization protocol was optimized and interference of organic solvents such as methanol was tested. For the immobilized AChE, limit of detection 2.75 ppb for aflatoxin B1 was received. The immobilized AChE kept full activity at least one month. Suitability of the assay for a practical performance is considered.     

Lactose Hydrolysis by β-Galactosidase Covalently Immobilized to Thermally Stable Biopolymers

Lactose has been hydrolyzed using covalently immobilized β-galactosidase on thermally stable carrageenan coated with chitosan (hydrogel). The hydrogel’s mode of interaction was proven by Fourier transform infrared spectroscopy, differential scanning calorimetry (DSC), and Schiff’s base formation. The DSC thermogram proved the formation of a strong polyelectrolyte complex between carrageenan and chitosan followed by glutaraldehyde as they formed one single peak. The modification of carrageenan improved the gel’s thermal stability in solutions from 35 °C to 95 °C. The hydrogel has been proven to be efficient for β-galactosidase immobilization where 11 U/g wet gel was immobilized with 50% enzyme loading capacity. Activity and stability of free and immobilized β-galactosidase towards pH and temperature showed marked shifts in their optimum pH from 4.5–5 to 5–5.5 and temperature from 50 °C to 45–55 °C after immobilization, which reveals higher catalytic activity and reasonable stability at wider pHs and temperatures. The apparent K _m of the immobilized enzyme increased from 13.2 to 125 mM, whereas the V _max increased from 3.2 to 6.6 μmol/min compared to the free enzyme, respectively. The free and immobilized enzymes showed lactose conversion of 87% and 70% at 7 h, respectively. The operational stability showed 97% retention of the enzyme activity after 15 uses, which demonstrates that the covalently immobilized enzyme is unlikely to leach. The new carrier could be suitable for immobilization of other industrial enzymes.