Immobilization of Acetylcholinesterase onto Pyrrole-containing Photocured Thermosets

Year 2023, , 95 - 102, 25.04.2023

Khadija Khaled Alı , Serap Gürbüzdemir , Emrah Çakmakçı , Ayşe Ogan

https://doi.org/10.19113/sdufenbed.1149256

Abstract

Acetylcholinesterase (AChE; EC 3.1.1.7) is a group of enzymes that catalyzes the hydrolysis of the neurotransmitter acetylcholine (ACh) into choline and acetate. AChE inhibition is commonly utilized as a biomarker for pesticides. In membrane based AChE biosensors the enzyme immobilization onto an electrode surface is of prime importance. In previous studies, conducting polymers-based supports have been used for the immobilization of AChE. In this study, a novel immobilization platform was developed. The simultaneous polymerization of pyrrole and functional thiol/ene monomers was performed to prepare conductive thermosets. AchE was covalently immobilized onto the membranes through the epoxy functional groups. After the immobilization process, the optimal temperature increased to 50 °C, displaying a better thermal stability and the optimum pH was elevated to 8.5. The activity of the immobilized enzyme was tested in the presence of several metals, and it was found that Cu2+ ions caused a noticable inhibition. After 10 cycles, the immobilized enzyme retained 51% of its original activity. In accordance with our results; the durability and the stability of the immobilized enzyme were improved. In future studies, the method applied here can be used in the design of an AchE biosensor.

Keywords

Acetylcholinesterase, Thiol-ene, Photocuring, Enzyme immobilization, Pyrrole

Supporting Institution

Marmara Üniv. BAP

Project Number

FEN-C-YLP-131217-0673

Asetilkolinesterazın Pirol İçeren Fotokürlenmiş Termosetlere İmmobilizasyonu

Abstract

Asetilkolinesteraz (AChE; EC 3.1.1.7), nörotransmiter asetilkolinin (ACh) kolin ve asetata hidrolizini katalize eden bir grup enzimdir. AChE inhibisyonu, pestisitler için biyolojik bir belirteç olarak yaygın olarak kullanılmaktadır. Membran temelli AChE biyosensörlerinde, elektrot yüzeyine enzimin immobilizasyonu oldukça önemlidir. AChE'nin immobilizasyonu için iletken polimerlerl de içeren pek çok farklı destek kullanılmıştır. Bu çalışmada, yeni bir immobilizasyon platformu geliştirilmiştir. Pirol ve tiyol-en monomerleri ile birlikte polimerleştirilerek iletken polimerik termosetler elde edilmesi planlanmıştır. AChE epoksi grupları üzerinden membrana immobilize edilmiştir. İmmobilizasyon işleminden sonra enzimin optimum sıcaklığı 50°C’ye çıkarak daha iyi bir termal kararlılık gösterirken, optimum pH değeri 8.5’a yükselmiştir. İmmobilize enzimin aktivitesi üzerine çeşitli metallerin etkisi araştırılmış ve Cu+2 iyonlarının önemli bir inhibisyona yol açtığı bulunmuştur. 10 döngüden sonra, immobilize enzimin başlangıç aktivitesinin %51'ine halen sahip olduğu belirlenmiştir. Sonuçlarımızla uyumlu olarak immobilize edilen enzimin kararlılığının ve dayanıklılığının arttığı görülmektedir. Gelecek çalışmalarda burada önerilen metod bir AChE biyosensör tasarımında kullanılabilir.

Keywords

Asetilkolinesteraz, Tiyol-en, Fotokürlenme, Enzim İmmobilizasyonu, Pirol

Project Number

FEN-C-YLP-131217-0673

References

• [1] Massoulié, J., Pezzementi, L., Bon, S., Krejci, E., Vallette, F. M. 1993. Molecular and Cellular Biology of Cholinesterases. Progress in neurobiology, 41(1), 31-91.
• [2] Pohanka, M. 2011. Cholinesterases, a Target of Pharmacology and Toxicology. Biomedical Papers of the Medical Faculty of Palacky University in Olomouc, 155(3).
• [3] Colovic, M. B., Krstic, D. Z., Lazarevic-Pasti, T. D., Bondzic, A. M., Vasic, V. M. 2013. Acetylcholinesterase Inhibitors: Pharmacology and Toxicology. Current neuropharmacology, 11(3), 315-335.
• [4] Karalliedde, L. 1999. Organophosphorus Poisoning and Anaesthesia. Anaesthesia, 54(11), 1073-1088.
• [5] Andreescu, S., Marty, J. L. 2006. Twenty Years Research in Cholinesterase Biosensors: from Basic Research to Practical Applications. Biomolecular engineering, 23(1), 1-15.
• [6] Martín-Barreiro, A., de Marcos, S., Jesús, M., Grazú, V., Galbán, J. 2018. Gold Nanocluster Fluorescence as an Indicator for Optical Enzymatic Nanobiosensors: Choline and Acetylcholine Determination. Sensors and Actuators B: Chemical, 277, 261-270.
• [7] Bodur, O. C., Özkan, E. H., Çolak, Ö., Arslan, H., Sarı, N., Dişli, A., Arslan, F. 2021. Preparation of acetylcholine biosensor for the diagnosis of Alzheimer's disease. Journal of Molecular Structure, 1223, 129168.
• [8] Jangi, S. R. H., Akhond, M. 2022. Introducing a Covalent Thiol-based Protected Immobilized Acetylcholinesterase with Enhanced Enzymatic Performances for Biosynthesis of Esters. Process Biochemistry, 120, 138-155.
• [9] D'souza, S. F. 1999. Immobilized Enzymes in Bioprocess. Current Science, 69-79.
• [10] Haider, T., Husain, Q. 2007. Calcium Alginate Entrapped Preparations of Aspergillus Oryzae β Galactosidase: Its Stability and Applications in the Hydrolysis of Lactose. International Journal of Biological Macromolecules, 41(1), 72-80.
• [11] Özkan, E. H., Yetim, N. K., Tümtürk, H., Sarı, N. 2015. Immobilization of acetylcholinesterase on Pt (II) and Pt (IV) attached nanoparticles for the determination of pesticides. Dalton Transactions, 44(38), 16865-16872.
• [12] Hasanoğlu Özkan, E., Kurnaz Yetim, N., Nartop, D., Sarı, N. 2021 Ensuring traceability of organophosphate pesticides (OPs) through enzyme immobilized spheres. Journal of the Iranian Chemical Society, 18(7), 1749-1759.
• [13] Nartop, D., Hasanoğlu Özkan, E., Yetim, N. K., Sarı, N. 2020. Qualitative enzymatic detection of organophosphate and carbamate insecticides. Journal of Environmental Science and Health, Part B, 55(11), 951-958.
• [14] Karmaz, E., Özkan, E. H., Yetim, N. K., Sarı, N. B. 2021. New Nanospheres to Use in the Determination of Imidan Phosmet and Vantex Pesticides. Journal of Inorganic and Organometallic Polymers and Materials, 31(7), 2915-2924.
• [15] Yetim, N. K., Özkan, E. H., Özcan, C., Sarı, N. 2020. Preparation of AChE immobilized microspheres containing thiophene and furan for the determination of pesticides by the HPLC-DAD method. Journal of Molecular Structure, 1222, 128931.
• [16] Asmussen, S., Arenas, G., Vallo, C. 2015. Photopolymerization of Pyrrole/Methacrylate Mixtures using α-Cleavage Type Photoinitiators in Combination with Iodonium Salt. Synthetic Metals, 209, 304-312.
• [17] Nazar, R., Ronchetti, S., Roppolo, I., Sangermano, M., Bongiovanni, R. M. 2015. In situ Synthesis of Polymer Embedded Silver Nanoparticles via Photopolymerization. Macromolecular Materials and Engineering, 300(2), 226-233.
• [18] Heydarnezhad, H. R., Pourabbas, B. 2013. One-Step Synthesis of Conductive Ceria/Polypyrrole Nanocomposite Particles via Photo-İnduced Polymerization Method. Journal of Materials Science: Materials in Electronics, 24(11), 4378-4385.
• [19] Ijeri, V. S., Nair, J. R., Gerbaldi, C., Gonnelli, R. S., Bodoardo, S., Bongiovanni, R. M. 2010. An Elegant and Facile Single-Step UV-Curing Approach to Surface Nano-Silvering of Polymer Composites. Soft Matter, 6(19), 4666-4668.
• [20] Razza, N., Blanchet, B., Lamberti, A., Pirri, F. C., Tulliani, J. M., Bozano, L. D., Sangermano, M. 2017. UV‐printable and Flexible Humidity Sensors based on Conducting/Insulating Semi‐Interpenetrated Polymer Networks. Macromolecular Materials and Engineering, 302(10), 1700161.
• [21] Bradford, M. M. 1976. A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-dye Binding. Analytical biochemistry, 72(1-2), 248-254.
• [22] Çakıroğlu, B., Çiğil, A. B., Ogan, A., Kahraman, M. V., Demir, S. 2018. Covalent Immobilization of Acetylcholinesterase on a Novel Polyacrylic Acid‐Based Nanofiber Membrane. Engineering in Life Sciences, 18(4), 254-262.
• [23] Ellman, G. L., Courtney, K. D., Andres Jr, V., Featherstone, R. M. 1961. A New and Rapid Colorimetric Determination of acetylcholinesterase activity. Biochemical pharmacology, 7(2), 88-95.
• [24] Ali, K.K. 2018. Immobilization of acetylcholinesterase on different polymeric surfaces. Marmara University, Institute for Graduate Studies in Pure and Applied Sciences, Master Science Thesis, 74s, Istanbul.
• [25] Aires-Trapote, A., Tamayo, A., Rubio, J., Rumbero, A., Hernáiz, M. J. 2015. Sustainable Synthesis of N-Acetyllactosamine using an Immobilized Β-Galactosidase on a Tailor Made Porous Polymer. RSC Advances, 5(50), 40375-40383.
• [26] Stoilova, O., Manolova, N., Gabrovska, K., Marinov, I., Godjevargova, T., Mita, D. G., Rashkov, I. 2010. Electrospun Polyacrylonitrile Nanofibrous Membranes Tailored for Acetylcholinesterase Immobilization. Journal of bioactive and compatible polymers, 25(1), 40-57.
• [27] Stoilova, O., Ignatova, M., Manolova, N., Godjevargova, T., Mita, D. G., Rashkov, I. 2010. Functionalized Electrospun Mats from Styrene–Maleic Anhydride Copolymers for Immobilization of Acetylcholinesterase. European Polymer Journal, 46(10), 1966-1974.
• [28] Kahraman, M. V., Bayramoğlu, G., Kayaman-Apohan, N., Güngör, A. 2007. α-Amylase immobilization on functionalized glass beads by covalent attachment. Food Chemistry, 104(4), 1385-1392.
• [29] Saleem, M., Rafiq, M., Seo, S. Y., Lee, K. H. 2016. Acetylcholinesterase Immobilization and Characterization, and Comparison of the Activity of the Porous Silicon-immobilized Enzyme with its Free Counterpart. Bioscience Reports, 36(2).
• [30] Yong, Y., Bai, Y. X., Li, Y. F., Lin, L., Cui, Y. J., Xia, C. G. 2008. Characterization of Candida Rugosa Lipase Immobilized onto Magnetic Microspheres with Hydrophilicity. Process Biochemistry, 43(11), 1179-1185.
• [31] Bayramoglu, G., Tunali, Y., Arica, M. Y. 2007. Immobilization of β-Galactosidase onto Magnetic Poly (GMA–MMA) Beads for Hydrolysis of Lactose in Bed Reactor. Catalysis Communications, 8(7), 1094-1101.
• [32] Amini, N., Mazinani, S., Ranaei-Siadat, S. O., Kalaee, M. R., Hormozi, S., Niknam, K., Firouzian, N. 2013. Acetylcholinesterase Immobilization on Polyacrylamide/Functionalized Multi-walled Carbon Nanotube Nanocomposite Nanofibrous Membrane. Applied biochemistry and biotechnology, 170(1), 91-104.
• [33] Ebadi, S. V., Fakhrali, A., Ranaei-Siadat, S. O., Gharehaghaji, A. A., Mazinani, S., Dinari, M., Harati, J. 2015. Immobilization of Acetylcholinesterase on Electrospun Poly (acrylic acid)/Multi-walled Carbon Nanotube Nanofibrous Membranes. RSC Advances, 5(53), 42572-42579.
• [34] Gabrovska, K., Nedelcheva, T., Godjevargova, T., Stoilova, O., Manolova, N., Rashkov, I. 2008. Immobilization of Acetylcholinesterase on New Modified Acrylonitrile Copolymer Membranes. Journal of Molecular Catalysis B: Enzymatic, 55(3-4), 169-176.
• [35] Moradzadegan, A., Ranaei‐Siadat, S. O., Ebrahim‐Habibi, A., Barshan‐Tashnizi, M., Jalili, R., Torabi, S. F., Khajeh, K. 2010. Immobilization of Acetylcholinesterase in Nanofibrous PVA/BSA Membranes by Electrospinning. Engineering in Life Sciences, 10(1), 57-64.
• [36] Yetim, N. K., Sarı, N. 2019. Preparation of ferrocene core dendrimers and immobilization of AChE for detection of diclofop-methyl herbicide. Macedonian Journal of Chemistry and Chemical Engineering, 38(2), 215–225.