The influence of the support nature on the kinetics parameters, inhibition constants and reactivation of immobilized acetylcholinesterase

Gabrovska, Katya; Marinov, I.; Godjevargova, Tzonka; Portaccio, Marianna; Lepore, Maria; Grano, V.; Diano, Nadia; Mita, D.G.

doi:10.1016/j.ijbiomac.2008.07.006

Cited by 42 publications

(21 citation statements)

References 46 publications

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“…The nitrile groups of the PAN‐based membranes were modified to form amino groups. Then, aldehyde derivatives were introduced to the membranes through the reaction with glutaraldehyde . The produced aldehyde groups could react specifically with the alpha‐amino residues of enzymes, which meant that the enzymes were covalently bonded to the PAN ultrafiltration membranes.…”

Section: Resultsmentioning

confidence: 99%

Immobilization of horseradish peroxidase on modified PAN‐based membranes for the removal of phenol from buffer solutions

Wang

Liu

Zheng³

et al. 2016

Can J Chem Eng

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Polyacrylonitrile ultrafiltration membranes have been widely used in many separation processes. In this paper, horseradish peroxidase was immobilized onto polyacrylonitrile ultrafiltration membrane by crosslinking with glutaraldehyde. The immobilized enzyme possessed a protein loading of 0.025 mg/cm2membrane and a specific activity of 105 U/mgprotein (105 μmol/min/mgprotein). The initial modified and immobilized membranes were observed using SEM and FTIR. Potential applications of HRP membranes were investigated in the removal of phenol through oxidation with the addition of hydrogen peroxide. The optimum pH of the immobilized enzyme was determined to be 6.0, and the optimum hydrogen peroxide concentration to be 30 mmol/L. Almost 100 % removal of phenol (1 ∼ 10 mg/L) from water was achieved by HRP membranes. For high concentrated solutions, successive cycles were successfully used to improve the degree of phenol oxidation. Furthermore, the immobilized horseradish peroxidase was operationally stable. These results suggest that the HRP membrane has promising applications for the removal of phenol.

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Section: Resultsmentioning

confidence: 99%

Immobilization of horseradish peroxidase on modified PAN‐based membranes for the removal of phenol from buffer solutions

Wang

Liu

Zheng³

et al. 2016

Can J Chem Eng

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“…K M value seems to be strongly affected by immobilization process as was also reported by Gabrovska et al where K M value of immobilized AChE was approximately 3 mM compare to 0.9 mM of free AChE [31] or it could be influenced by external processes like irradiation as was described by Barteri et al who calculated K M value to 1.37 mM [32]. A high value of K M was also provided by human mutant AChE [33,34].…”

Section: Resultsmentioning

confidence: 67%

Construction of an Acetylcholinesterase Sensor Based on Synthesized Paramagnetic Nanoparticles, a Simple Tool for Neurotoxic Compounds Assay

Kostelník

Kopel

Cegan

et al. 2017

Sensors

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Magnetic particles (MPs) have been widely used in biological applications in recent years as a carrier for various molecules. Their big advantage is in repeated use of immobilized molecules including enzymes. Acetylcholinesterase (AChE) is an enzyme playing crucial role in neurotransmission and the enzyme is targeted by various molecules like Alzheimer’s drugs, pesticides and warfare agents. In this work, an electrochemical biosensor having AChE immobilized onto MPs and stabilized through glutaraldehyde (GA) molecule was proposed for assay of the neurotoxic compounds. The prepared nanoparticles were modified by pure AChE and they were used for the measurement anti-Alzheimer’s drug galantamine and carbamate pesticide carbofuran with limit of detection 1.5 µM and 20 nM, respectively. All measurements were carried out using screen-printed sensor with carbon working, silver reference, and carbon auxiliary electrode. Standard Ellman’s assay was used for validation measurement of both inhibitors. Part of this work was the elimination of reversible inhibitors represented by galantamine from the active site of AChE. For this purpose, we used a lower pH to get the original activity of AChE after inhibition by galantamine. We also observed decarbamylation of the AChE-carbofuran adduct. Influence of organic solvents to AChE as well as repeatability of measurement with MPs with AChE was also established.

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“…A higher K M value means lower affinity between the enzyme and substrate, which could be due to the introduction of steric hindrance created by the support towards the active site, as well as by the lack of sufficient enzyme flexibility for binding of the natural ligands during catalysis. 27,29,32 Additionally, the V MAX values evaluated for HuGAPDH-IMER are approximately 6-fold higher than those exhibited for the free enzyme (Table 1). Although it is difficult to estimate the extent of the derivatization with glutaraldehyde onto the capillary, the increase in the V MAX values might be attributed to the presence of a large number of potential reaction sites for covalent coupling with the protein, thereby increasing the amount of active enzyme immobilized onto the capillary.…”

Section: Kinetics Parametersmentioning

confidence: 85%

Structural insights into the molecular basis responsible for the effects of immobilization on the kinetic parameters of glyceraldehyde-3-phosphate dehydrogenase from Trypanosoma cruzi and human

Güido¹,

Cardoso²,

Moraes³

et al. 2010

J. Braz. Chem. Soc.

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O desenvolvimento de métodos rápidos e eficazes para a identificação de novas moléculas bioativas é fundamental para o processo de descoberta e planejamento de fármacos. A integração de um sistema de cromatografia liquida de alta eficiência (CLAE), com biorreatores como fase estacionária (IMER) é uma estratégia atrativa e versátil para a triagem de coleções de compostos visando à identificação de novos agentes terapêuticos. Os parâmetros cinéticos da enzima imobilizada gliceraldeído-3-fosfato desidrogenase (GAPDH) de Trypanosoma cruzi e humana foram determinados (T. cruzi:; humana:) e comparados com aqueles observados em solução (T. cruzi:). Os resultados indicaram uma diminuição na afinidade das enzimas imobilizadas, entretanto, os elementos estruturais necessários para o processo de reconhecimento molecular e atividade biológica permaneceram inalterados, aumentando a estabilidade das enzimas. Além disso, a análise estrutural forneceu dados moleculares importantes envolvidos nos efeitos da imobilização sobre as interações entre ligante e receptor e, consequentemente, sobre a atividade enzimática e parâmetros cinéticos.The development of fast and reliable methods for the identification of new bioactive compounds is of utmost importance to boost the process of drug discovery and development. Immobilized enzyme reactors (IMERs), integrated with high performance liquid chromatography (HPLC), are attractive and versatile tools for screening collections consisting of natural products and synthetic small molecules. Standard kinetic parameters of the immobilized enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from both Trypanosoma cruzi de and human have been determined (T. cruzi:; humana:), and comparisons of these values with those of the parasite and human free enzymes indicate a decrease in the affinity for the immobilized system (T. cruzi:). Interestingly, despite the kinetic differences between the two systems, the immobilized GAPDHs retained the required structural requirements for molecular recognition and biological activity, increasing the stability the enzyme. In the present work, we described an integrated structural analysis which has provided important insights into the molecular basis underlying the effects of immobilization on the ligand-receptor interactions and consequent enzymatic activity and kinetics parameters.

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The influence of the support nature on the kinetics parameters, inhibition constants and reactivation of immobilized acetylcholinesterase

Cited by 42 publications

References 46 publications

Immobilization of horseradish peroxidase on modified PAN‐based membranes for the removal of phenol from buffer solutions

Immobilization of horseradish peroxidase on modified PAN‐based membranes for the removal of phenol from buffer solutions

Construction of an Acetylcholinesterase Sensor Based on Synthesized Paramagnetic Nanoparticles, a Simple Tool for Neurotoxic Compounds Assay

Structural insights into the molecular basis responsible for the effects of immobilization on the kinetic parameters of glyceraldehyde-3-phosphate dehydrogenase from Trypanosoma cruzi and human

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