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Reactivity of adenosine triphosphatase adsorbed on nitrocellulose
2001
Momic, T. | Banjac, A. | Horvat, A. (Institut za nuklearna istrazivanja Vinca, Beograd (Yugoslavia). Laboratorija za molekularnu biologiju)
Possibilities of application of transmembrane enzymes as a biological component of a biosensor for water quality control and detection of toxical substances were performed. Synaptic plasma membranes (SPMs) were adsorbed on nitrocellulose filters. The adsorption of SPMs was followed by determination of the transmembrane enzyme Na,K-ATPase. The optimal conditions for SPM adsorption on nitrocellulose filters were determined: 25 microgram per nitrocellulose filter disc during 1 hour of incubation, on - 20 deg C. The ATPase activity of adsorbed SPM showed, that almost 30% of enzymic activity was detected on nitrocellulose filters in mentioned conditions. This results showed that adsorption of SPM on solid support enhancing enzymatic stability and enable its industrial and analytical application.
Показать больше [+] Меньше [-]Application of laccase immobilized rice straw biochar for anthracene degradation
2021
Imam, Arfin | Suman, Sunil Kumar | Singh, Raghuvir | Vempatapu, Bhanu Prasad | Ray, Anjan | Kanaujia, Pankaj K.
The present study explores the immobilization of ligninolytic enzyme-laccase on the surface of rice straw biochar and evaluates its application for anthracene biodegradation. The rice straw biochar was acid-treated to generate carboxyl functionality on its surface, followed by detailed morphological and chemical characterization. The surface area of functionalized biochar displayed a two-fold increase compared to the untreated biochar. Laccase was immobilized on functionalized biochar, and an immobilization yield of 66% was obtained. The immobilized enzyme demonstrated operational stability up to six cycles while retaining 40% of the initial activity. Laccase immobilization was further investigated by performing adsorption and kinetic studies, which revealed the highest immobilization concentration of 500 U g⁻¹ at 25 °C. The adsorption followed the Langmuir isotherm model at equilibrium, and the kinetic study confirmed pseudo-second-order kinetics. The equilibrium rate constant (K₂) at 25 °C and 4 °C were 3.6 × 10⁻³ g U⁻¹ min⁻¹ and 4 × 10⁻³ g U⁻¹ min⁻¹ respectively for 100 U g⁻¹ of enzyme loading. This immobilized system was applied for anthracene degradation in the aqueous batch mode, which resulted in complete degradation of 50 mg L⁻¹ anthracene within 24 h of interaction exposure.
Показать больше [+] Меньше [-]Effect of the immobilized microcystin-LR-degrading enzyme MlrA on nodularin degradation and its immunotoxicity study
2020
Wu, Xiang | Wu, Hao | Gu, Xiaoxiao | Zhang, Rongfei | Sheng, Qiang | Ye, Jinyun
In freshwater ecosystems with frequent cyanobacterial blooms, the cyanobacteria toxin pollution is becoming increasingly serious. Nodularin (NOD), which has strong biological toxicity, has emerged as a new pollutant and affects the normal growth, development and reproduction of aquatic organisms. However, little information is available regarding this toxin. In this study, a graphene oxide material modified by L-cysteine was synthesized and used to immobilize microcystin-LR (MC-LR)-degrading enzyme (MlrA) to form an immobilized enzyme nanocomposite, CysGO-MlrA. Free-MlrA was used as a control. The efficiency of NOD removal by CysGO-MlrA was investigated. Additionally, the effects of CysGO-MlrA and the NOD degradation product on zebrafish lymphocytes were detected to determine the biological toxicity of these two substances. The results showed the following: (1) There was no significant difference in the degradation efficiency of NOD between CysGO-MlrA and free-MlrA; the degradation rate of both was greater than 80% at 1 h (2) The degradation efficiency of the enzyme could retain greater than 81% of the initial degradation efficiency after the CysGO-MlrA had been reused 7 times. (3) CysGO-MlrA retained greater than 50% of its activity on the 8th day when preserved at 0 °C, while free-MlrA lost 50% of its activity on the 4th day. (4) CysGO-MlrA and the degradation product of NOD showed no obvious cytotoxicity to zebrafish lymphocytes. Therefore, CysGO-MlrA might be used as an efficient and ecologically safe degradation material for NOD.
Показать больше [+] Меньше [-]Non-covalent assembled laccase-graphene composite: Property, stability and performance in beta-blocker removal
2019
Immobilization of enzymes on carriers have been pursued to make the enzyme stable, reusable and obtaining even better enzyme activity. Due to the highly stable two-dimensional layer structure, large surface area and pore volume, graphene materials were seemed as ideal carrier for enzyme immobilization. In this paper, pristine few layer graphene (FLG) was applied to interact with laccase to synthesize laccase-graphene composite and the results of AFM, FT-IR and adsorption isotherm suggested that laccase was loaded on the FLG with a very high loading dosage (221.1 mg g⁻¹). Based on the measured interaction force and binding type between laccase and graphene, we proposed that the great enzyme loading on FLG is likely due to the non-covalent π-π stacking in addition to the large surface area of FLG. The composite has better stability to the variance of pH and storage temperature than free laccase. The synthesized composite can effectively transform beta-blocker labetalol with an enhanced efficiency, though the possible reaction pathways kept not changing. We further performed molecular simulation study on the crystal structure variation of laccase binding on FLG and proposed that catalytic activity enhancement may be attributed to the more exposure extent of the catalytic center of laccase. In addition, the laccase-graphene composite can be reused more than ten times in catalyzing the labetalol removal.
Показать больше [+] Меньше [-]Biotransformation of disperse dyes using nitroreductase immobilized on magnetic particles modified with tosyl group: Identification of products by LC-MS-MS and theoretical studies conducted with DNA
2018
Franco, Jefferson Honorio | Silva, Bianca F. da | de Castro, Alexandre A. | Ramalho, Teodorico C. | Pividori, María Isabel | Zanoni, Maria Valnice Boldrin
The present work evaluates the action of nitroreductase enzyme immobilized on Tosylactivated magnetic particles (MP-Tosyl) on three disperse dyes which contain nitro and azo groups. The dyes included Disperse Red 73 (DR 73), Disperse Red 78 (DR 78), and Disperse Red 167 (DR 167). The use of a magnet enabled the rapid and easy removal of the immobilized enzyme after biotransformation; this facilitated the identification of the products generated using high-performance liquid chromatography with diode array detector (HPLC-DAD) and mass spectrometry (LC-MS/MS). The main products formed by the in vitro biotransformation were identified as the product of nitro group reduction to the correspondent amine groups, which were denoted as follows: 50% of 2-(2-(4-((2-cyanoethyl)(ethyl)amino)phenyl)hydrazinyl)-5-nitrobenzonitrile, 98% of 3-((4-((4-amino-2-chlorophenyl) diazenyl)phenyl) (ethyl)amino)propanenitrile and 99% of (3-acetamido-4 - ((4-amino-2-chlorophenyl) diazenyl) phenyl) azanediyl) bis (ethane-2,1-diyl) for DR 73, DR 78 and DR 167, respectively. Based on the docking studies, the dyes investigated were found to be biotransformed by nitroreductase enzyme due to their favorable interaction with the active site of the enzyme. Theoretical results show that DR73 dye exhibits a relatively lower rate of degradation; this is attributed to the cyanide substituent which affects the electron density of the azo group. The docking studies also indicate that all the dyes presented significant reactivity towards DNA. However, Disperse Red 73 was found to exhibit a substantially higher reactivity compared to the other dyes; this implies that the dye possesses a relatively higher mutagenic power. The docking results also show that DR 73, DR 78 and DR 167 may be harmful to both humans and the environment, since the mutagenicity of nitro compounds is associated with the products formed during the reduction of nitro groups. These products can interact with biomolecules, including DNA, causing toxic and mutagenic effects.
Показать больше [+] Меньше [-]Functionalized Materials as a Versatile Platform for Enzyme Immobilization in Wastewater Treatment
2021
Kołodziejczak-Radzimska, Agnieszka | Nghiem, Long D. | Jesionowski, Teofil
PURPOSE OF REVIEW: Untreated wastewater discharge can significantly and negatively impact the state of the environment. Rapid industrialization and economic development have directly contributed to land and water pollution resulting from the application of many chemicals such as organic dyes, pharmaceuticals, and industrial reagents. The removal of these chemicals before effluent discharge is crucial for environmental protection. This review aims to explore the importance of functionalized materials in the preparation of biocatalytic systems and consider their application in eliminating water pollutants. RECENT FINDINGS: Wastewater treatment methods can be classified into three groups: (i) chemical (e.g., chemical oxidation and ozonation), (ii) physical (e.g., membrane separation and ion exchange), and (iii) biological processes. Biological treatment is the most widely used method due to its cost-effectiveness and eco-friendliness. In particular, the use of immobilized enzymes has recently become more attractive as a result of scientific progress in advanced material synthesis. The selection of an appropriate support plays an important role in the preparation of such biologically active systems. Recent studies have demonstrated the use of various materials for enzyme immobilization in the purification of water. This review identifies and discusses different biocatalytic systems used in the enzymatic degradation of various water pollutants. Materials functionalized by specific groups can serve as good support matrices for enzyme immobilization, providing chemical and thermal stability to support catalytic reactions. Enzymatic biocatalysis converts the pollutants into simpler products, which are usually less toxic than their parents. Due to immobilization, the enzyme can be used over multiple cycles to reduce the cost of wastewater treatment. Future studies in this field should focus on developing new platforms for enzyme immobilization in order to improve degradation efficiency.
Показать больше [+] Меньше [-]Impacts of horseradish peroxidase immobilization onto functionalized superparamagnetic iron oxide nanoparticles as a biocatalyst for dye degradation
2022
Keshta, Basem E. | Gemeay, Ali H. | Khamis, Abeer A.
To enhance the dye removal efficiency by natural enzyme, horseradish peroxidase (HRP) was immobilized onto amine-functionalized superparamagnetic iron oxide and used as a biocatalyst for the oxidative degradation of acid black-HC dye. The anchored enzyme was characterized by vibrating sample magnetometry, Fourier transform infrared spectroscopy, X-ray diffraction, thermogravimetry, scanning electron microscopy, Brunauer–Emmett–Teller and Barrett–Joyner–Halenda methods, nitrogen adsorption–desorption measurements, Zeta potential, energy dispersive X-ray spectroscopy, and transmission electron microscopy. The Michaelis constant values of free and immobilized HRP were determined to be 4.5 and 5 mM for hydrogen peroxide and 12.5 and 10 mM for guaiacol, respectively. Moreover, the maximum values of free and immobilized HRP were 2.4 and 2 U for H₂O₂, respectively, and 1.25 U for guaiacol. The immobilized enzyme was thermally stable up to 60°C, whereas the free peroxidase was stable only up to 40°C. In the catalytic experiment, the immobilized HRP exhibited superior catalytic activity compared with that of free HRP for the oxidative decolorization and removal of acid black-HC dye. The influence of experimental parameters such as the catalyst dosage, pH, H₂O₂ concentration, and temperature on the removal efficiency was investigated. The reaction followed second-order kinetics, and the thermodynamic activation parameters were determined.
Показать больше [+] Меньше [-]Production of hydroxytyrosol rich extract from Olea europaea leaf with enhanced biological activity using immobilized enzyme reactors
2022
As olive leaves constitute the main by-product of the olive oil industry with important environmental and economic impact, there is an increasing demand for its valorization. In the present work, we report the development and application of immobilized enzyme batch bioreactors for the chemo-enzymatic treatment of an aqueous Olea europaea leaf extract rich in oleuropein to produce an extract enriched in hydroxytyrosol and other oleuropein hydrolysis products. To this end, a robust biocatalyst was developed through the immobilization of β-glucosidase on chitosan-coated magnetic beads which exhibited high hydrolytic stability after 240 h of incubation at 37 °C. The biocatalyst was successfully used in both a rotating bed-reactor and a stir-tank reactor for the modification of the olive leaf extract leading to high conversion yields of oleuropein (exceeding 90%), while an up to 2.5 times enrichment in hydroxytyrosol was achieved. Over 20 phenolic compounds (from different classes of phytochemicals such as flavonoids, secoiridoids, and their derivatives) were identified, in the extract before and after its modification through various chromatographic and spectroscopic techniques. Finally, the biological activity of both extracts was evaluated. Compared to the non-modified extract, the modified one demonstrated 20% higher antioxidant activity, seven-fold higher antibacterial activity, and enhanced cytotoxicity against leiomyosarcoma cells.
Показать больше [+] Меньше [-]Immobilization of horseradish peroxidase on Fe3O4 nanoparticles for enzymatic removal of endocrine disrupting chemicals
2020
Xiao, Feng | Xiao, Ping | Jiang, Wei | Wang, Dongsheng
The modified Fe₃O₄ nanoparticles were used as a support for the immobilization of horseradish peroxidase (HRP). The immobilized enzyme (HRP@Fe₃O₄) was characterized by transmission electron microscopy (TEM), X-ray diffraction (XRD), Fourier-transform infrared spectrometer (FTIR), and vibration sample magnetometer (VSM). According to the results, the optimum concentration of glutaraldehyde (GA) and agitation time were 300 μL and 7 h. HRP was well loaded on the surface of the Fe₃O₄. There was no change in the crystal structure of HRP@Fe₃O₄ compared with Fe₃O₄. The removals of bisphenol A (BPA) and 17α-ethinylestradiol (EE2) using HRP@Fe₃O₄ had been investigated. The degradation efficiencies of BPA and EE2 catalyzed by HRP@Fe₃O₄ were higher than that of soluble HRP. In addition, HRP@Fe₃O₄ can be reused through magnetic separation. After the fifth repeated use, the removal efficiencies of BPA and EE2 were up to 56% and 48%, respectively. Batch studies of catalyzed oxidation and coagulation on the degradation of BPA and EE2 in the presence of humic acid (HA) were also investigated. The order of the removal efficiencies was HRP+PACl (polyaluminum chloride)+SDS (lauryl sodium sulfate)>HRP+PACl>HRP>HRP+PAM (Polyacrylamide)>HRP+PAM+SDS. The coagulation effect of HRP@Fe₃O₄ and PACl was better than that of HRP@Fe₃O₄ and PAM. The removals of BPA and EE2 were 90.3% and 64.5% by use HRP@Fe₃O₄ and PACl as coagulant, while the removals were 78.7% and 57.6% by use HRP@Fe₃O₄ and PAM as coagulant. SDS had a positive effect on PACl, while a negative effect on PAM. Moreover, the products generated by enzymatic oxidation reaction can be effectively removed after coagulation.
Показать больше [+] Меньше [-]Immobilization of Carbonic Anhydrase on Chitosan Stabilized Iron Nanoparticles for the Carbonation Reaction
2012
Yadav, Renu | Joshi, Meenal | Wanjari, Snehal | Prabhu, Chandan | Kotwal, Swati | Satyanarayanan, T. | Rayalu, Sadhana
Carbonic anhydrase (CA) has been immobilized on chitosan stabilized iron nanoparticles (CSIN) for the biomimetic carbonation reaction. CSIN was characterized using scanning electron microscope, energy dispersive X-ray, X-ray diffraction spectroscopy, and Fourier transform infrared analysis. The effect of various parameters such as pH, temperature and storage stability, on immobilized CA was investigated using a p-NPA assay. Kinetic parameters of immobilized and free CA (K ₘ and V ₘₐₓ values) were also evaluated. The K ₘ and V ₘₐₓ for immobilized CA was 1.727 mM and 1.189 μmol min⁻¹ ml⁻¹, respectively, whereas for free enzyme the K ₘ and V ₘₐₓ was 1.594 mM and 1.307 μmol min⁻¹ ml⁻¹, respectively. It was observed that the immobilized enzyme had longer storage stability and retained 50 % of its initial activity upto 30 days at room temperature. CA immobilized on CSIN has been used for hydration of CO₂, and the results were validated by using a gas chromatographic method. Proof of concept has been established for the biomimetic carbonation reaction. Immobilized CA show reasonably good CO₂ sequestration capacity of 21.55 mg of CaCO₃/mg of CA as compared to CO₂ sequestration capacity of 34.92 mg of CaCO₃/mg of CA for free CA respectively, under a limiting concentration of CO₂ (14.5 mg of CO₂/10 ml).
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