Stasyuk N. Microbial enzymes combined with nanozymes for creation of bio(chemo)sensors and test kits for determining the content of practically important analytes

Українська версія

Thesis for the degree of Doctor of Science (DSc)

State registration number

0524U000002

Thesis Registration Form

0524U000002.pdf

Applicant for

Specialization

  • 03.00.07 - Мікробіологія

17-01-2024

Specialized Academic Board

Д 35.246.01

Institute of Cell Biology of the National Academy of Sciences of Ukraine

Essay

The presented work is devoted to a comprehensive study of the structural, physicochemical, and catalytic properties of nanozymes in combination with microbial enzymes, as well as to the study of the biological and toxic effects (in vivo) of nanozyme composites on Ogataea (Hansenula) polymorpha yeast cells. The main focus of the work is directed at the conjugation of nanomaterials with microbial enzymes and on their basis the creation of new bio(chemo)sensors and test kits for determining the content of practically important analytes, in particular, creatinine, L-arginine, glucose L-lactate, ethanol, and methyl amine. Experimental data were obtained which, for the first time in the world, prove the possibility of replacing the natural enzyme catalase with artificial "catazyme", which restores the ability of mutant non-catalase yeast cells to grow on methanol. Molecular-genetic analysis and sequencing of the genome locus of the yeast strain O. polymorpha C-105 revealed two mutations in the catalase gene: replacement of nitrogenous base of cytosine (C) at the 895 position of the coding sequence by thymine (T) and deletion of C at the 896 position; this combination leads to a shift in the reading frame and the formation of the stop codon TGA, which causes the production of non-functional, incomplete catalase protein in the strain C-105. The different cytotoxic effect of synthesized nanozymes on the yeast O. polymorpha cells was shown for the first time. For the first time, a combination of catalytic nanomaterials and highly purified preparations of enzymes of hydrolases, in particular, arginase I, creatinine deaminase, and arginine deaminase was used in the formation of bioselective elements of amperometric biosensors selective for L-arginine and creatinine. New enzyme-nanozyme methods for ethanol analysis were created for the first time using nanomimetics of peroxidase and natural alcohol oxidase.

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Research papers

1) Stasyuk N, Zakalskiy A, Nogala W, Gawinkowski S, Ratajczyk T, Bonarowska M, Demkiv O Zakalska O, Gonchar M (2023) A reagentless amperometric biosensor for creatinine assay based on recombinant creatinine deiminase and N-methylhydantoin-sensitive CoCu nanocomposite. Sensors and Actuators B: Chemical 393: 34276. https://doi.org/10.1016/j.snb.2023.134276

2) Prokopiv T, Stasyuk N, Gonchar M (2023) Nanozyme can substitute a natural Ogataea polymorpha catalase enzyme in vivo. Microchimica Acta 190(5): 174. doi: 10.1007/s00604-023-05753-8

3) Stasyuk N, Demkiv O, Gayda G, Zakalska O, Nogala W, Gonchar M (2022) Amperometric biosensors based on alcohol oxidase and peroxidase-like nanozymes for ethanol assay. Microchimica Acta 189: 474. doi: 10.1007/s00604-022-05568-z

4) Stasyuk N, Gayda G, Kavetskyy T, Gonchar M (2022) Nanozymes with reductase-like activities: electrochemical and antioxidant properties. RSC Advances 12: 2026 – 2035. doi: 10.1039/d1ra08127f

5) Stasyuk N, Demkiv O, Gayda G, Zakalska O, Zakalskiy A, Serkiz R, Kavetskyy T, Gonchar M (2022) Reusable alcohol oxidase-nPtCu/alginate beads for highly sensitive ethanol assay in beverages. RSC Advances 12: 21309 – 21317. doi: 10.1039/d2ra02106d

6) Stasyuk N, Demkiv O, Gayda G, Zakalskiy A, Klepach H, Bisko N, Gonchar M, Nisnevitch M (2022) Highly Porous 3D Gold Enhances Sensitivity of Amperometric Biosensors Based on Oxidases and CuCe Nanoparticles. Biosensors 12: 472. doi: 10.3390/bios12070472

7) Stasyuk NYe, Gayda GZ, Zakalskiy AE, Fayura LR, Zakalska OM, Sibirny АА, Nisnevitch M, Gonchar MV (2022) Amperometric biosensors for L-arginine and creatinine assay based on recombinant deiminases and ammonium-sensitive Cu/Zn(Hg)S nanoparticles. Talanta 238 (1): 122996, https://doi.org/10.1016/j.talanta.2021.122996

8) Stasyuk N, Gayda G, Demkiv O, Darmohray L, Gonchar M, Nisnevitch M (2021) Amperometric biosensors for L-arginine determination based on L-arginine oxidase and peroxidase-like nanozymes. Applied Sciences 11(15): 7024. https://doi.org/10.3390/app11157024

9) Demkiv O, Stasyuk N, Serkiz R, Gayda G, Nisnevitch, M, Gonchar M (2021) Peroxidase-Like Metal-Based Nanozymes: Synthesis, Catalytic Properties, and Analytical Application. Applied Science 11: 777. https://doi.org/10.3390/app11020777

10) Zakalskiy A, Stasyuk N, Zakalska O, Boretsky Y, Gonchar M (2020) Overexpression and one-step renaturation-purification of the tagged creatinine deiminase of Corynebacterium glutamicum in Escherichia coli cells. Cell Biology International 44 (5), 1204–1211. https://doi.org/10.1002/cbin.11320

11) Stasyuk N, Gayda G, Zakalskiy A, Zakalska O, Serkiz R, Gonchar M (2019) Amperometric biosensors based on oxidases and PtRu nanoparticles as artificial peroxidase. Food Chemistry 285: 213-220. https://doi.org/10.1016/j.foodchem.2019.01.117

12) Stasyuk N, Gayda G, Zakalskiy A, Zakalska O, Errachid A, Gonchar M (2018) Highly selective apo-arginase based method for sensitive enzymatic assay of manganese (II) and cobalt (II) ions. Spectrochimica Acta, Part A: Molecular and Biomolecular Spectroscopy 193, 349 – 356. doi: 10.1016/j.saa.2017.12.031

13) Stasyuk N, Gayda G, Yepremyan H, Stepien A, Gonchar M (2017) Fluorometric enzymatic assay of L-arginine. Spectrochimica Acta, Part A: Molecular and Biomolecular Spectroscopy 193 170: 184-190. https://doi.org/10.1016/j.saa.2016.07.019

14) Stasyuk N, Gayda G, Zakalskiy A, Zakalska O, Fayura L, Vovk O, Stasyk O, Sibirny A, Gonchar M (2017) Recombinant Forms of Arginase and Arginine Deiminase as Catalytic Components of Argitest Enzymatic Kit. Science and Innovation 13(4): 56—63. https://doi.org/10.15407/scine13.04.056

15) Stasyuk N, Gayda G, Klepach H, Semashko T, Gonchar M (2017) Nanoparticles of Noble Metals for Enzymatic Sensors: An Amperometric Glucose Biosensor for Wine Analysis. Sensors Letters 15(8), 647 – 654. https://doi.org/10.1166/sl.2017.3862

16) Stasyuk N, Gayda G, Fayura L, Boretskyy Y, Gonchar M, Sibirny A (2016) Novel arginine deiminase-based method to assay L-Arginine in beverages. Food Chemistry 201: 320-326. https://doi.org/10.1016/j.foodchem.2016.01.093

17) Stasyuk N, Gayda G, Serkiz RJa, Gonchar M (2016) The “green” synthesis of gold nanoparticles by the yeast Hansenula polymorpha. Вісник Львівського університету. Серія біологічна. 73: 96 – 102. http://nbuv.gov.ua/UJRN/VLNU_biol_2016_73_18

18) Stasyuk N, Gayda G, Klepach H, Gonchar M (2016) Nanoparticles of noble metals as effective platforms for the fabrication of amperometric biosensor on hydrogen peroxide. Sensors Letters 14: 1169 – 1179 https://doi.org/10.1166/sl.2016.3748

19) Karkovska M, Smutok O, Stasyuk N, Gonchar M (2015) L-Lactate-selective microbial sensor based on flavocytochrome b2-enriched yeast cells using recombinant and nanotechnology approaches, Talanta 144, 1195–1200. https://doi.org/10.1016/j.talanta.2015.07.081

20) Stasyuk NYe, Bass S. R., Gayda G. Z., Yepremyan H. S., Gonchar MV (2015) New enzymatic method for L-arginine assay based on human arginase I and urease. ScienceRise 6 (1), 43-48. https://journals.uran.ua/sciencerise/article/view/45126

21) Stasyuk NYe, Gayda GZ, Serkiz RJa, Gonchar MV (2015) Cell Imaging with Fluorescent Bi-Metallic Nanoparticles. Journal of Advances in Chemistry 11 (4): 3499 – 3511. https://rajpub.com/index.php/jac/article/view/6694

22) Stasyuk N, Gayda G, Gonchar M (2014) L-arginine-selective microbial amperometric sensor based on recombinant yeast cell over-producing human liver arginase I. Sensors and Actuators B: Chemical 204: 515-521. https://doi.org/10.1016/j.snb.2014.06.112

23) Stasyuk NYe, Smutok OV, Zakalskiy AE, Zakalska OM, Gonchar MV (2014) Methylamine-sensitive amperometric biosensor based on (His)6-tagged Hansenula polymorpha methylamine oxidase immobilized on the gold nanoparticles. BioMed Research International 2014: 1-8. doi: 10.1155/2014/480498

24) Synenka MM, Stasyuk NYe, Semashko TV, Gayda GZ, Mikhailova RV, Gonchar MV (2014) Immobilization of oxidoreductases at/on gold and silver nanoparticles. Studia Biologica 8 (3-4): 5–12. http://publications.lnu.edu.ua/journals/index.php/biology/article/view/192

25) Патент України на корисну модель №142490 Україна, МПК G01N21/64 Моноензиматичний спосіб визначення вмісту креатиніну та амонію в біологічних рідинах людини / Гончар М.В., Закальський А.Є., Закальська О.М., Стасюк Н.Є., Прокопів Т.М., Борецький Ю.Р., Демків О.М.; заявник і власник Інститут біології клітини НАН України, Львівський державний університет фізичної культури імені Івана Боберського – № u201911544, заявл. 29.11.2019, опубл. 10.06.2020, Бюл. №11. – 8 с. [патент України на корисну модель]

26) Патент України на корисну модель №107543 Україна, МПК G01N 21/64 Ензиматичний метод визначення L-аргініну в крові людини / Стасюк Н.Є., Гайда Г.З., Закальський А.Є., Закальська О.М., Гончар М.В.; заявник і власник Інститут біології клітини НАН України – № u201512716, заявл. 22.12.2015, опубл. 10.06.2016, Бюл. №11. – 5 с. [патент України на корисну модель]

27) Патент на корисну модель №108773 Україна, МПК G01N 33/52, G01N 21/64 Ензиматично-хімічний метод визначення вмісту L-аргініну в харчових продуктах та алкогольних напоях / Стасюк Н.Є., Гайда Г.З., Фаюра Л.Р., Борецький Ю.Р., Сибірний А.А., Гончар М.В.; заявник і власник Інститут біології клітини НАН України – № u201601804, заявл. 25.02.2016, опубл. 25.07.2016, Бюл. №14. – 7 с. [патент України на корисну модель]

28) Gayda G, Stasyuk N, Zakalskiy A, Gonchar M, Katz E (2022) Arginine-hydrolyzing enzymes for electrochemical biosensors. Сurrent Opin Electrochem. 33: 100941. https://www.sciencedirect.com/science/article/abs/pii/S2451910322000060

29) Stasyuk N, Smutok O, Demkiv O, Prokopiv T, Gayda G, Nisnevitch M, Gonchar M. (2020) Synthesis, Catalytic Properties and Application in Biosensorics of Nanozymes and Electronanocatalysts: A Review. Sensors, 20 (16): 4509. https://www.mdpi.com/1424-8220/20/16/4509

30) Zakalskiy A., Stasyuk N., Gonchar M. (2019) Creatinine Deiminase: Characterization, Using in Enzymatic Creatinine Assay, and Production of the Enzyme. Curr Protein Pept Sci. 20: 465-470 https://pubmed.ncbi.nlm.nih.gov/30426898/

31) Gayda G, Stasyuk N, Serkiz R, Gonchar M. Fluorescent nanoparticles of noble metals for cell imaging (ред. Н.А.Серебрякова). Київ: НВП «Видавництво «Проект наукова книга»; 2017. С. 156 – 165. http://ir.librarynmu.com/handle/123456789/7658

32) Карковська М, Стасюк Н., Гайда Г., Смуток О., Гончар М. Наноматеріали у конструюванні біосенсорів медичного призначення (ред. Н.А.Серебрякова). Київ: НВП «Видавництво «Проект наукова книга»; 2017. С. 165 – 177. http://ir.librarynmu.com/handle/123456789/7658

33) Gonchar M, Smutok O, Karkovska M, Stasyuk N, Gayda G. Yeast-based biosensors for clinical diagnostics and food control (Ed. А.А. Sibirny). Springer International Publishing AG "Biotechnology of Yeasts and Filamentous Fungi"; 2017. P. 392 – 400. https://dokumen.pub/non-conventional-yeasts-from-basic-research-to-application-1st-ed-2019-978-3-030-21109-7-978-3-030-21110-3.html

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