Synthesıs of novel hydrazone derıvatıves, screenıng of theır free radıcal scavengıng actıvıtıes and evaluatıon of theır in vitro ınhıbıtory effects on purıfıed human serum paraoxonase-1

Bu çalışmada, dört yeni hidrazon türevi sentezlenmiş ve yapıları X-Işını (XRD), Nükleer Manyetik Rezonans (H-NMR), Fourier Dönüşümlü Infrared Spektrofotometre (FTIR), Ultraviyole-Görünür spektrofotometre (UV-VIS) taramaları ve elementel analiz teknikleri ile aydınlatılmıştır. Tüm yeni sentezlenmiş bileşikler, 2,2-difenil-1-picrylhydrazyl (DPPH) yöntemiyle serbest radikal giderme aktiviteleri için taramaya tabi tutulmuştur. En düşük IC50 değerine (0,185 mg/mL) sahip B-2,4-MBPyH bileşiği, DPPH için en yüksek serbest radikal giderme aktivitesi göstermiştir. Paraoksonaz1 (PON1: EC 3.1.8.1) yüksek yoğunluklu lipoproteinlere (HDL) sıkıca bağlı, yapısında kalsiyum içeren bir metalo enzimdir ve memelilerde düşük yoğunluklu lipoproteinlerin (LDL) oksidasyonuna karşı koruyucu etkisi bulunmaktadır. Bu çalışmada, insan serum paraoksonaz1 (hPON1) enzimi, iki-aşamalı yöntem ile; amonyum sülfat çöktürmesi ve Sepharose-4B-L-tirozin-1-naftilamin hidrofobik etkileşim kromatografisi kullanılarak saflaştırılmıştır. Saflaştırılan enzimin SDS-poliakrilamid jel elektroforezinde, 43 kDa molekül ağırlığında tek bir band göstermiştir. Saflaştırılan enzim 21,22 U/mg spesifik aktiviteye sahiptir. Kullanılan yöntem ile % 17,484 verim ve 561,375 saflaştırma derecesine ulaşılmıştır. Ayrıca, paraokson substrat olarak kullanılarak, saflaştırılmış enzimin Km ve Vmax değerleri sırayla 0,018496 mM ve 114,96 U/mL olarak belirlenmiştir. Bu çalışmada, sentezlenen yeni bileşiklerin saflaştırılmış hPON1 üzerine in vitro inhibisyon etkileri de incelenmiştir. Sonuçlar, tüm hidrazin türevlerinin hPON1 enzim aktivitesini derişime bağlı olarak inhibe ettiğini göstermiştir. Çalışılan hidrazon türevleri arasında, en düşük IC50 değerine (0,0138 mg/mL) sahip B-2,3-MBPyH bileşiğinin hPON1 aktivitesi için en etkin inhibitör olduğu bulunmuştur. Bu çalışma, hPON1 aktivitesinin, çalışılan hidrazon türevlerine karşı oldukça yüksek derecede hassas olduğunu göstermiştir.In this study, four new hydrazone derivatives were synthesized and their structures have been elucidated by X-Ray Diffraction (XRD), Nuclear Magnetic Resonance (H-NMR), Fourier transform infrared spectroscopy (FTIR), ultraviolet-visible spectrophotometry (UV-VIS) scanning and elemental analysis techniques. All the newly synthesized compounds were subjected to screening for their free radical scavenging activity by 2,2-diphenyl-1-picrylhydrazyl (DPPH) method. Compound B-2,4-MBPyH with the lowest IC50 value of (0.185 mg/mL) showed the highest free radical scavenging activity of DPPH. Paraoxonase-1 (PON1: EC 3.1.8.1) is a calcium-dependent enzyme associated with high-density lipoproteins (HDLs) and has a protective effect against oxidation of low-densitylipoproteins (LDLs) in mammals. In this study, human serum paraoxonase1 (hPON1) was purified using two-step procedures, namely ammonium sulphate precipitation and Sepharose-4B-L-tyrosine-1-naphthylamine hydrophobic interaction chromatography. SDS–polyacrylamide gel electrophoresis of the purified enzyme showed a single band with an apparent MW of 43 kDa. The purified enzyme had a specific activity of 21.22 U/mg. The overall purification fold and yield were found to be % 17.484 and 561.375 respectively. Furthermore, using the paraoxon as a substrate, we determined the Km and Vmax values of the purified enzyme, as 0.018496 mM and 114.955 U/mL, respectively. In this study, in vitro inhibition effect of these synthesized novel compounds on purified hPON1 were also investigated by using paraoxon as a substrate. The results showed that all the hydrazone derivatives inhibited the hPON1 enzyme activity in a concentration-dependent fashion. Among the studied hydrazone derivatives, B-2,3-MBPyH was found to be the most effective inhibitor for hPON1 activity, with the lowest IC50 values of (0.0138 mg/mL). The present study has demonstrated that hPON1 activity is very highly sensitive to studied hydrazone derivatives

Synthesis, characterization and biological assessment of a novel hydrazone as potential anticancer agent and enzyme inhibitor

Noma, Samir Abbas Ali ( Aksaray, Yazar ) Erzengin, Mahmut ( Aksaray, Yazar )In present work, a novel hydrazone compound, namely 1-(5-bromo-2,3-dimethoxybenzylidene)-2-(pyridine-2-yl) hydrazine (5Br2DM2PH), was synthesized by condensation reaction in acidic conditions. The structure of this compound was detailed elucidated by 1H NMR, FT-IR, UV/VIS and XRD methods. Antioxidant activity of the 5Br2DM2PH compound was investigated by scavenging activity on 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals. The 5Br2DM2PH compound exhibited high scavenging activity compared with reference antioxidant. To determine in vitro cytotoxic effect of its, cell viability was tested using MTT assay on human breast (MCF-7) and colon (Caco-2) cancer cell lines. The results showed that the cytotoxic effect of the 5Br2DM2PH is more potent than cisplatin. The IC50 value for MCF-7 is 16.8 μM while the value for Caco-2 is 11.8 μM. In addition, the in vitro inhibitory property of the compound 5Br2DM2PH was carried out on human paraoxonase-1 (hPON-1) and the IC50 value was found as 13.8 μg. Our ambitions for the novel 5Br2DM2PH compound will guide many future studies in the field of medicine, analytical and drug applications, as well as new organic synthesis

Development of L-asparaginase@hybrid Nanoflowers (ASNase@HNFs) Reactor System with Enhanced Enzymatic Reusability and Stability

Hybrid nanoflowers materials have recently received great attention in enzyme immobilization applications because of the advantages such as their large surface area, excellent stability, simple, eco-friendly, and cost-effective synthesis. In this study, l-asparaginase which is an important commercial enzyme in the medicine and food industry was selected as a model enzyme. To the best of our knowledge, this study is the first report of designing L-asparaginase@hybrid nanoflowers to enhance its enzymatic performance. L-asparaginase@hybrid nanoflowers were synthesized using ASNase as an organic component and Cu(II) ion as inorganic component. They were characterized by their morphology and chemical point of view by using different techniques. The synthesized L-asparaginase@hybrid nanoflowers exhibited high residual activity at broad pH and high temperature ranges in comparison to free form. Moreover, L-asparaginase@hybrid nanoflowers possessed good reusability and excellent long-time storage stability. Especially, L-asparaginase@hybrid nanoflowers-3 maintained nearly 51 and 75% of its original activity, respectively, after nine consecutive catalytic cycles and storage at 30 degrees C for 4 weeks. The results indicated that these hybrid nanoflowers will be promising carrier matrix for the immobilization of ASNase in biotechnological applications with improved catalytic properties

A New Design to Enhance the Enzyme Activities: Investigation of L-Asparaginase Catalytic Performance by IMAC Effect on g-C3N4 Nanolayers

Recently, graphite carbon nitride (g-C3N4) has come to the fore as a new material with its carbon-based two-dimensional structure, simple preparation procedure, and excellent physicochemical stability properties. This study aims to investigate the activity and kinetic studies of the L-asparaginase enzyme via immobilized metal ion affinity chromatography (IMAC) process of g-C3N4 nanolayers. Firstly, g-C3N4 nanolayers were synthetized and Ni2+ ions were binded their surfaces. The synthesized samples were investigated by SEM, ICP-MS, XRD, and FTIR. The highest L-ASNase adsorption on Ni2+-g-C3N4 nanostructures was 444.1 mg/g, at 3 mg/mL L-ASNase concentration. Optimal medium conditions for L-ASNase adsorption occurred at pH 8.0 and 25 °C. The immobilized enzyme showed improved stability relating to the soluble enzyme in extreme situations. On the other hand, the storage stability and reusability of the immobilized enzyme were found to be approximately 64 and 53% of the original activity after 29 days at room temperature and 10 cycles, respectively. From the Michaelis–Menten constants Km and Vmax, both of them decreased after immobilization compare to the free one. The obtained outcomes showed that the g-C3N4 is a suitable matrix for L-asparaginase immobilization with ideal catalytic efficiency and improved stability

L-asparaginase immobilized p(HEMA-GMA) cryogels: A recent study for biochemical, thermodynamic and kinetic parameters

*Önal, Burcu ( Aksaray, Yazar ) *Odabaşı, Mehmet ( Aksaray, Yazar )Cryogels have recently been attracted intense attention as suitable carriers for enzyme immobilization. Herein, L-asparaginase was selected as the model enzyme due to its application such as pharmaceutical and food. Under optimum conditions, L-asparaginase was immobilized on poly (2-hydroxyethyl methacrylate-glycidyl methacrylate) cryogels with 68.8% of immobilization yield and 69.3% of activity recovery. The immobilized enzyme exhibited improved stability with respect to the soluble enzyme at extreme conditions, especially around acidic pH and high temperature. Also, the storage stability and reusability of the immobilized enzyme were found to be approximately 54% and 52% of the original activity after 28 days at room temperature and 10 cycles, respectively. The thermodynamic studies indicated that activation energy (E-a) of the free enzyme decreased from 13.08 to 10.97 kJ/mol, which means an increase in the thermostability of L-asparaginase. The Michaelis-Menten constants (K-m) of 2.04 and 1.67 mM, and the maximum reaction rates (V-max) of 170.0 and 115.0 mu M min(-1) were estimated for soluble and immobilized L-asparaginase, respectively. These findings demonstrated that the designed cryogels turn out to be a good carrier matrix for L-asparaginase immobilization with high catalytic efficiency and enhanced stability

New 2-hydroxyethyl substituted N-Heterocyclic carbene precursors: Synthesis, characterization, crystal structure and inhibitory properties against carbonic anhydrase and xanthine oxidase

Here, the synthesis, spectral and the structural studies of 2-hydroxyethyl substituted N-heterocyclic carbene (NHC) precursors and the enzyme inhibition activities of the NHC precursors were investigated against the cytosolic carbonic anhydrase I and II isoenzymes (hCA I and hCA II), and xanthine oxidase (XO). The IC50 values of NHC precursors against these enzymes were determined by spectrophotometric method. The spectra of new NHC precursors have been obtained by using H-1 NMR, C-13 NMR, FTIR spectroscopy and elemental analysis techniques. The structure of a new NHC precursor was established by using single-crystal X-ray diffraction method. The results of inhibition experiment indicated that all 2-hydroxyethyl substituted NHC derivatives showed remarkable inhibition activity toward hCA I, hCA II and XO. The range of IC50 values for hCA I, hCA II and XO inhibition was determined as 0.1565-0.5127, 0.1524-0.5368 and 1.253-5.342 mu M. Especially, trimethylbenzyl derivative of 2-hydroxyethyl substituted NHC precursor has demonstrated high inhibition effect on all studied enzymes due to steric bulk of this substituent. (C) 2019 Elsevier B.V. All rights reserved

A rational approach for 3D recognition and removal of L-asparagine via molecularly imprinted membranes

In this study, a L-asparagine (L-Asn) imprinted membranes (L-Asn-MIPs) were synthesized via molecular imprinting for selective and efficient removal of L-Asn. The L-Asn-MIP membrane was prepared by using acrylamide (AAm) and hydroxyethyl methacrylate (HEMA) as a functional monomer and a comonomer, respectively. The membrane was characterized by scanning electron microscopy (SEM) and Fourier Transform infrared spectroscopy (FTIR). The L-Asn adsorption capacity of the membrane was investigated in detail. The maximum L-Asn adsorption capacity was determined as 408.2 mg/g at pH: 7.2, 24 °C. Determination of L-Asn binding behaviors of L-Asn-MIPs also shown with Scatchard analyses. The effect of pH on L-Asn adsorption onto the membrane and also the selectivity and reusability of the L-Asn-MIPs for L-Asn adsorption were determined through L-asparaginase (L-ASNase) enzyme activity measurements. The selectivity of the membrane was investigated by using two different ternary mixtures; L-glycine (L-Gly)/L-histidine (L-His)/L-Asn and L-tyrosin (L-Tyr)/L-cystein(L-Cys)/L-Asn. The obtained results showed that the L-Asn-MIP membranes have a high selectivity towards L-Asn

Synthesis and characterization of Near-Infrared (NIR)-light triggerable upconverting nanoparticles to enhance activity of immobilized l-asparaginase

In this study, PEG-L-ASNase enzyme was immobilized on UCNPs and its activity was improved by NIR. NaYF4: Yb3+, Er3+/PEI UCNPs were characterized to observe structural and morphological changes before/after enzyme immobilization. Additionally, optimum pH and optimum temperature values were determined for PEG-L-ASNase and NaYF4: Yb3+, Er3+/PEI-PEG-L-ASNase. While the optimum pH value was found to be 8.5 and 8 for PEG-L-ASNase and NaYF4: Yb3+, Er3+/PEI-PEG-L-ASNase, the optimum temperature values were determined as 40 oC and 45 oC, respectively. Also, after incubation at 50 °C for 6 hours, it was observed that PEG-L-ASNase and NaYF4: Yb3+, Er3+/ PEI-PEG-L-ASNase retained 28% and 59% of their initial activities, respectively. Moreover, the kinetic parameters (Km and Vmax) were calculated. These values for PEG-L-ASNase were found as 2.31 ± 0.04 mM and 140.85 ± 3.23 μmol/min, respectively. For NaYF4: Yb3+, Er3+/PEI-PEG-L-ASNase, the Km and Vmax values were calculated as 1.56 ± 0.06 mM and 138.89 ± 1.66 μmol/min, respectively. Finally, it was determined that the application of NIR at 800 mW, for 60 min at 1 cm increased the activity approximately 4 times compared to the PEG-L-ASNase. As a result, L-ASNase was successfully immobilized and the activity of the immobilized enzyme was increased approximately 4 times by using NIR light. Also, in this study is one of the pioneering studies in terms of the development of triggerable drug delivery systems.No sponso

Comparative study of ASNase immobilization on tannic acid-modified magnetic Fe3O4/SBA-15 nanoparticles to enhance stability and reusability

*Acet, Ömür ( Aksaray, Yazar ) *Odabaşı, Mehmet ( Aksaray, Yazar )In this work, l-asparaginase was immobilized on tannic acid-modified magnetic mesoporous particles. In brief, Fe3O4/SBA-15/tannic acid magnetic particles were synthesized, and their structures and morphologies were fully characterized using various methods. The properties of the free and immobilized enzyme were examined in terms of pH, temperature, thermal stability, storage stability, and reusability. Moreover, the effects of metal ions, inhibitors and organic solvents on the activity of the immobilized enzyme were investigated. Compared to the free enzyme, the immobilized enzyme possessed better tolerance to changes in ambient temperature and pH. Additionally, thermal incubation results showed that the free enzyme lost its activity, while the immobilized enzyme exhibited the opposite behavior. Most strikingly, the immobilized l-asparaginase exhibited a high degree of activity (70%) after being reused 16 times while also demonstrating 71% and 63% storage stability of the initial activity even after 28 days at 4 °C and room temperature, respectively. Together with these results, l-asparaginase was successfully immobilized upon Fe3O4/SBA-15/tannic acid magnetic nanoparticles with improved stability properties. This support holds great potential and opens up a novel perspective for growing applications