Electrochemical study of catalytic activity of  Erwinia carotovora  L-asparaginase immobilized on bacterial cellulose films

Alina Berezhnova; Anastasia Shishparenok; Lyubov Agafonova; Dmitry Zhdanov; Victoria Shumyantseva

doi:10.15826/chimtech.10354

Electrochemical study of catalytic activity of Erwinia carotovora L-asparaginase immobilized on bacterial cellulose films

Alina Berezhnova, Anastasia Shishparenok, Lyubov Agafonova, Dmitry Zhdanov, Victoria Shumyantseva

Abstract

In this work, we developed an electrochemical method for the determination of the catalytic activity of L-ASNase from Erwinia carotovora noncovalently immobilized by means of physical adsorption on bacterial cellulose (BC) films (BC/L-ASNase). Immobilization of enzymes on a support can refine stability and catalytic activity. Determination of enzyme on BC and catalytic activity evaluation of its immobilized form is based on the electrochemical oxidation of amino acids from L-ASNase active center and polypeptide backbones. The electrochemical behavior of L-ASNase on screen-printed electrodes modified with single-wall carbon nanotubes (SPE/CNTs) was studded. In the presence of the substrate L-asparagine, the oxidation current of the enzyme decreased in a concentration-dependent manner. The sigmoidal dependence of maximum peal current on substrate concentration was registered with parameters K_0.5 = 800 µM and Hill positive coefficient as n = 3. The sensitivity, LOD and LOQ for L-asparagine were calculated as 0.89 μA/mM, 0.10 mM, and 0.28 mM.

Keywords

Erwinia carotovora L-asparaginase; bacterial cellulose; electrochemistry; screen-printed electrode; carbon nanotubes; L-asparagine

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References

Basso A, Serban S. Industrial Applications of Immobilized Enzymes—A Review. Mol. Catal. 2019;479:110607. doi:10.1016/j.mcat.2019.110607

Bernal C, Rodriguez K, Martinez R. Integrating Enzyme Immobilization and Protein Engineering: An Alternative Path for the Development of Novel and Improved Industrial Biocatalysts. Biotechnol. Adv. 2018;36(5):1470-1480. doi:10.1016/j.biotechadv.2018.06.002

Sheldon RA, Woodley JM. Role of Biocatalysis in Sustainable Chemistry. Chem. Rev. 2018;118(2):801–838. doi:10.1021/acs.chemrev.7b00203

Romero-Fernandez M., Paradisi F. General Overview on Immobilization Techniques of Enzymes for Biocatalysis. In Catalysts Immobilization. New York: John Wiley & Sons; 2019. 409-435 pp.

O’Connell A, Barry A, Burke AJ, Hutton AE, Bell EL, Green AP, O’Reilly E. Biocatalysis: Landmark Discoveries and Applications in Chemical Synthesis. Chem. Soc. Rev. 2024;53(6):2828-2850. doi:10.1039/d3cs00689a

Rossino G, Robescu MS, Licastro E, Tedesco C, Martello I, Maffei L, Vincenti G, Bavaro T, Collina S. Biocatalysis: A Smart and Green Tool for the Preparation of Chiral Drugs. Chirality. 2022;34(11):1403-1418. doi:10.1002/chir.23498

Liang JF, Li YT, Yang VC. Biomedical application of immobilized enzymes. J Pharm Sci. 2000;89(8):979-90. doi:10.1002/1520-6017(200008)89:8%3C979::AID-JPS2%3E3.0.CO;2-H

Tosta Perez M, Herrera Belen L, Letelier P, Calle Y, Pessoa A, Farías JG. L-Asparaginase as the gold standard in the treatment of acute lymphoblastic leukemia: a comprehensive review. Med Oncol. 2023;40(5):150. doi:10.1007/s12032-023-02014-9

Kwon YM, Chung HS, Moon C, Yockman J, Park YJ, Gitlin SD, David AE, Yang VC. L-Asparaginase encapsulated intact erythrocytes for treatment of acute lymphoblastic leukemia (ALL). J Control Release. 2009;139(3):182-189. doi:10.1016/j.jconrel.2009.06.027

Heo YA, Syed YY, Keam SJ. Pegaspargase: A Review in Acute Lymphoblastic Leukaemia. Drugs. 2019;79(8):901. doi:10.1007/s40265-019-01137-6

Hanada K, Kawada K, Obama K. Targeting Asparagine Metabolism in Solid Tumors. Nutrients. 2025;17(1):179. doi:10.3390/nu17010179

Kislyak IA, Pokrovskaya MV, Zhanturina DY, Pokrovsky VS. The use of L-asparaginase for the treatment of solid tumors: data from experimental studies and clinical trials. Russian Journal of Oncology. 2023;28(1):79-94. doi:10.17816/onco562802

Shisparenok AN, Gladilina YuA, Pokrovskaya MV, Aleksandrova SS, Kraevsky SV, Ivanov YuD, Kozlov AF, Zlotnikov ID, Kudryashova EV, Atroshenko DL, Zhdanov DD. Immobilization of L-asparaginase on oxidized bacterial cellulose to enhance stability and cytotoxic activity. International Journal Biological Macromolecules. 2026;339(1):149992. doi: 10.1016/j.ijbiomac.2025.149992

Shisparenok AN, Koroleva SA, Dobryakova NV, Gladilina YA, Gromovykh TI, Solopov AB, Kudryashova EV, Zhdanov DD. Bacterial cellulose films for L-asparaginase delivery to melanoma cells. International Journal Biological Macromolecules. 2024;276:133932. doi: 10.1016/j.ijbiomac.2024.133932

Shisparenok AN, Petryaev ER, Koroleva SA, Dobryakova NV, Zlotnikov ID, Komedchikova EN, Kolesnikova OA, Kudryashova EV, Zhdanov DD. Bacterial Cellulose-Chitosan Composite for Prolonged-Action L-Asparaginase in Treatment of Melanoma Cells. Biochemistry Moscow. 2024;89(10):1727-1743. doi:10.1134/S0006297924100067

Vergov B, Sbirkov Y, Minchev D, Todorova T, Baldzhieva A, Burnusuzov H, Spasova МI, Sarafian V. Implementation of plate reader-based indooxine and Nessler protocols for monitoring L-asparaginase serum activity in childhood acute lymphoblastic leukaemia. Biol Methods Protoc. 2024;9(1):bpae042. doi:10.1093/biomethods/bpae042

Brown S, Muhamad N, and Simcock DC. Estimating enzyme kinetic parameters from apparent KMs and Vmaxs. World Academy of Science, Engineering and Technology. 2010;4:431-436.

Shumyantseva V, Bulko T, Pronina V, Kanashenko S, Pokrovskaya M, Aleksandrova S, Zhdanov D. Electroenzymatic model system for the determination of catalytic activity of Erwinia carotovora L-asparaginase. Processes. 2022;10(7):1313. doi:10.3390/pr10071313

Somji M, Dounin V, Muench SB, Schulze H, Bachmann TT, Kerman K. Electroanalysis of amino acid substitutions in bioengineered acetylcholinesterase. Bioelectrochemistry 2012;88:110-113. doi:10.1016/j.bioelechem.2012.07.004.

Papageorgiou AC, Posypanova, GA, Andersson CS, Sokolov NN, Krasotkina, J. Structural and functional insights into Erwinia carotovora L-asparaginase. FEBS J. 2008;275(17):4306-4316. doi:10.1111/j.1742-4658.2008.06574.x

Carrara S, Baj-Rossi C, Boero C, De Micheli G. Do Carbon Nanotubes contribute to Electrochemical Biosensing? Electrochimica Acta. 2014;128:102-112. doi:10.1016/j.electacta.2013.12.123

Ferrari AGM, Rowley-Neale SJ, Banks CE. Screen-printed electrodes: Transitioning the laboratory in-to-the field. Talanta Open. 2021;3:100032. doi:10.1016/j.talo.2021.100032

Compton BRG, Banks CE. Understanding Voltammetry. London: Imperial College Press; 2011.

Nicholson RS, Shain I. Theory of stationary electrode polarography. Single scan and cyclic methods applied to reversible, irreversible, and kinetic systems. Analyt. Chem. 1964;36:706-723.

Manna S, Sharma A, Satpati AK. Electrochemical methods in understanding the redox processes of drugs and biomolecules and their sensing. Curr. Opin. Electrochem. 2022;32:100886. doi:10.1016/j.coelec.2021.100886

Bard AJ, Faulkner LR. Electrochemical Methods: Fundamentals and Applications. New York: John Wiley & Sons Inc; 2001.

Borek D, Jaskólski M. Sequence analysis of enzymes with asparaginase activity. Acta Biochim Pol. 2001;48(4):893-902. doi:10.18388/abp.2001_3855

Borek D, Kozak M, Pei J, Jaskolski M. Crystal structure of active site mutant of antileukemic L-asparaginase reveals conserved zinc-binding site. .FEBS J. 2014;281(18):4097-111. doi:10.1111/febs.12906

Pokrovskaya MV, Pokrovsky VS, Aleksandrova SS, Sokolov NN, Zhdanov DD. Molecular Analysis of L-Asparaginases for Clarification of the Mechanism of Action and Optimization of Pharmacological Functions. Pharmaceutics. 2022;14(3):599. doi:10.3390/pharmaceutics14030599

Derst C, Wehner A, Specht V, Rohm KH. States and functions of tyrosine residues in Escherichia coli asparaginase II. Eur. J. Biochem. 1994;224(2):533-540. doi:10.1111/j.1432-1033.1994.00533.x

Aung HP, Bocola M, Schleper S, Röhm KH. Dynamics of a mobile loop at the active site of Escherichia coli asparaginase. Biochim Biophys Acta. 2000;1481(2):349-359. doi:10.1016/s0167-4838(00)00179-5

Lubkowski J, Vanegas JM, Chan WK, Lorenzi PL, Weinstein JN, Sukharev S, Fushman D, Rempe S, Anishkin A, Wlodawer A. Mechanism of Catalysis by L-asparaginase. Biochemistry. 2020;59(20):1927-1945. doi:10.1021/acs.biochem.0c00116

Lubkowski J, Wlodawer A. Geometric considerations support the double‐displacement catalytic mechanism of L‐asparaginase. Protein Sci. 2019;28(10):1850-1864. doi:10.1002/pro.3709

Lubkowski J, Wlodawer A. Structural and biochemical properties of L-asparaginase. FEBS J. 2021;288(14):4183-4209. doi:10.1111/febs.16042

Agrawal S, Jana UK, Kango N. Heterologous expression and molecular modelling of L-asparaginase from Bacillus subtilis ETMC-2. International Journal of Biological Macromolecules. 2021;192:28-37. doi:10.1016/j.ijbiomac.2021.09.186

Magri A, Soler MF, Lopes AM, Cilli EM, Barber PS, Pessoa JrA, Pereira JFB. A critical analysis of L-asparaginase activity quantification methods - colorimetric methods versus high-performance liquid Chromatography. Anal. Bioanal. Chem. 2018;410:6985-6990. doi:10.1007/s00216-018-1326-x

Alexandrova SS, Gladilina YA, Pokrovskaya MV, Sokolov NN, Zhdanov DD. Mechanisms of development of side effects and drug resistance to asparaginase and ways to overcome them. Biomed. Khim. 2022;16:175-186. doi:10.18097/PBMC20226802104

Krasotkina J, Borisova AA, Gervaziev YV, Sokolov NN. One-step purification and kinetic propertiesof the recombinant L-asparaginase from Erwinia carotovora. Biotechnol. Appl. Biochem. 2004;39(2):215-221. doi:10.1042/BA20030138

Rusling JF, Wang B, Yun S. Electrochemistry of redox enzymes. In Bioelectrochemistry: Fundametals, Experimental Techniques and Applications. New Jersey: John Wiley & Sons; 2008. 39-85 pp.

Masamrekh R, Kuzikov A, Veselovsky A, Toropygin I, Shkel T, Strushkevich N, Gilep A, Usanov S, Archakov A, Shumyantseva V. Interaction of 17α-hydroxylase, 17(20)-lyase (CYP17A1) inhibitors – abiraterone and galeterone – with human sterol 14α-demethylase (CYP51A1). J. Inorg. Biochem. 2018;186:24-33. doi:10.1016/j.jinorgbio.2018.05.010

Bisswanger H. Enzyme kinetics. Principles and methods. Germany: Wiley-VCH Verlag GmbH & Co. KGaA; 2008. 32 p.

Sokolov NN, Eldarov MA, Pokrovskaya MV, Aleksandrova SS, Abakumova OYu, Podobed OV, Melik-Nubarov NS, Kudryashova EV, Grishin DV, Archakov AI. Bacterial Recombinant L-Asparaginases: Properties, Structure, and Anti_Proliferative Activity. Biochem. Moscow Suppl. Ser. B: Biomedical Chemistry. 2015;9(4):325–338. doi:10.1134/S199075081504006X

Shishparenok AN, Blinova VG, Zhdanov DD. Fusion Protein Technology to Enhance Pharmacological Properties of L-Asparaginases. Biomolecules. 2026;16(7):963. doi:10.3390/biom16070963

Das R, Srivastava R, Rai S, Kayastha AM, Srivastava A, Kar RK, Singh C. Integrative simulation and spectroscopy illuminates L-asparaginase stability on nanocomposite surface for biosensing. International Journal of Biological Macromolecules. 2026;372:153082. doi:10.1016/j.ijbiomac.2026.153082

Vimal A, Kumar A. Biotechnological production and practical application of L-asparaginase enzyme. Biotechnol Genet Eng Rev. 2017;33(1):40-61. doi:10.1080/02648725.2017.1357294

Suresh SA, Ethiraj S, Rajnish KN. A systematic review of recent trends in research on therapeutically significant l-asparaginase and acute lymphoblastic leukemia. Mol. Biol. Rep. 2022;49(12):11281–11287. doi:10.1007/s11033-022-07688-4

Chi H, Jiang Q, Wang W, Niu J, Zhang G, Xu D, Lu Z, Zhu P, Lu F, Ran T. Structural basis and functional mining of active site loop for fine tuning substrate affinity of L-asparaginase from Bacillus licheniformis. Journal of Hazardous Materials. 2026:142189. doi:10.1016/j.jhazmat.2026.142189

Nayak BS, Samanta S, Singh S, Sonawane A. Emerging advancements to improve L-asparaginase-based therapy: New insight into old paradigm, a review. International Journal of Biological Macromolecules. 2025:149664. doi:10.1016/j.ijbiomac.2025.149664

Kumar K, Kataria M, Verma N. Plant asparaginase-based asparagine biosensor for leukemia. Artifi cial Cells, Nanomedicine, and Biotechnology. 2013;41(3):184-188. doi:10.3109/10731199.2012.716062

DOI: https://doi.org/10.15826/chimtech.10354

Copyright (c) 2026 Alina Berezhnova, Anastasia Shishparenok, Lyubov Agafonova, Dmitry Zhdanov, Victoria Shumyantseva

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