Noreen Nordin1, Rahmath Abdulla1, Siti Aqlima Ahmad2, and Mohd Khalizan Sabullah This email address is being protected from spambots. You need JavaScript enabled to view it.1

1Faculty Science and Natural Resources, Universiti Malaysia Sabah, 88400, Kota Kinabalu, Sabah, Malaysia
2Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia


Received: December 8, 2020
Accepted: May 10, 2021
Publication Date: October 12, 2021

 Copyright The Author(s). This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are cited.

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The continuous discharge of toxic materials into the environment has been an alarming issue faces around the globe. Hence, matching effort of monitoring activity is vital to coping with the overwhelming amount of metal ions. Along with the significant current research being conducted, this study aims to investigate the sensitivity of acetylcholinesterase (AChE) of Sabah porcupine fish, Diodon hystrix as an alternative biosensor in the detection of heavy metals. The enzyme was precipitated followed by the purification using ammonium sulfate precipitation and procainamide-affinity chromatography, respectively, with a total recovery of 66.67% with the specific activity of 2297.50 U/mg. The enzyme works optimally at pH 9 with the best incubation temperature of 30°C. The Michaelis constant (Km) and maximal velocity (Vmax) of 1.171 mM and 879257 mol/min/mg denotes the highest catalytic efficiency (Vmax/Km) of acetylthiocholine iodide (ATC) as its preferable substrate. Inhibition study tested on 10 metal ions resulted in increasing toxicity order of Cr6+ < Co2+ < Ag2+ < Cu2+ < Pb2+ < As5+ < Cd2+ < Zn2+ < Ni2+ < Hg2+, with only Hg2+ exhibited the half-maximal inhibitory concentration (IC50) of 0.48 mg/L. From the study, it suggests that the D. hystrix AChE as the potential conventional biosensor for heavy metals detection.

Keywords: Acetylcholinesterase; Diodon hystrix; Heavy metals; Pollution


  1. [1] L. C. M. Lebreton, J. Van Der Zwet, J.-W. Damsteeg, B. Slat, A. Andrady, and J. Reisser, (2017) “River plastic emissions to the world’s oceans" Nature communications 8(1): 1–10. DOI: 10.1038/ncomms15611.
  2. [2] Conserve Energy Future, (2019) “40 interesting facts about water pollution you’ll wish you’d known":
  3. [3] H. Ali, E. Khan, I. I. J. of Chemistry, and U. 2019, (2019) “Environmental chemistry and ecotoxicology of hazardous heavy metals: environmental persistence, toxicity, and bioaccumulation" Journal of Chemistry 2019: 1–14. DOI: 10.1155/2019/6730305.
  4. [4] WWF, (2017) “Integrated river basin management (IRBM)":
  5. [5] N. Khatri and S. Tyagi, (2015) “Influences of natural and anthropogenic factors on surface and groundwater quality in rural and urban areas" Frontiers in Life Science 8(1): 23–39. DOI: 10.1080/21553769.2014.933716.
  6. [6] J. N. Edokpayi, J. O. Odiyo, and O. S. Durowoju, (2017) “Impact of wastewater on surface water quality in developing countries: a case study of South Africa"Water quality: 401–416. DOI: 10.5772/66561.
  7. [7] M. Haseena, M. Malik, A. Javed, S. Arshad, et al., (2018) “Water pollution and human health" Environ Risk Assess Remediat 1(3): 16–19. DOI: 10.4066/2529-8046.100020.
  8. [8] S. Parimoolam and F. A. Dahalan, (2013) “Physicochemical optimization of granular sludge in rubber industrial wastewater" Journal of Biochemistry, Microbiology and Biotechnology 1(1): 7–10.
  9. [9] A. Basirun, S. Ahmad, N. Yasid, M. Sabullah, H. Daud, S. Sha’arani, A. Khalid, and M. Shukor, (2019) “Toxicological effects and behavioural and biochemical responses of Oreochromis mossambicus gills and its cholinesterase to copper: a biomarker application" International Journal of Environmental Science and Technology 16(2): 887–898. DOI: 10.1007/s13762-018-1711-1.
  10. [10] P. B. Tchounwou, C. G. Yedjou, A. K. Patlolla, and D. J. Sutton, (2012) “Heavy metal toxicity and the environment" Molecular, clinical and environmental toxicology 101: 133–164. DOI: 10.1007/978-3-7643-8340-4_6.
  11. [11] M. K. Sabullah, M. R. Sulaiman, M. S. Shukor, M. T. Yusof,W. L.W. Johari, M. Y. Shukor, and A. Syahir, (2015) “Heavy metals biomonitoring via inhibitive assay of acetylcholinesterase from Periophthalmodon schlosseri" Rendiconti Lincei 26(2): 151–158. DOI: 10.1007/s12210-014-0359-0.
  12. [12] N. M. Hayat, N. A. Shamaan, M. Y. Shukor, M. K. Sabullah, M. A. Syed, A. Khalid, F. A. Dahalan, K. A. Khalil, and S. A. Ahmad, (2015) “Cholinesterase-based biosensor using Lates calcarifer (Asian Seabass) brain for detection of heavy metals" J Chem Pharm Sci 8(2): 376–81. DOI: 10.22438/jeb/38/3/MRN-987.
  13. [13] N. M. Hayat, S. A. Ahmad, N. A. Shamaan, M. K. Sabullah, M. Y. A. Shukor, M. A. Syed, A. Khalid, K. A. Khalil, and F. A. Dahalan, (2017) “Characterisation of cholinesterase from kidney tissue of Asian seabass (Lates calcarifer) and its inhibition in presence of metal ions" Journal of Environmental Biology 38(3): 383. DOI: 10.22438/jeb/38/3/MRN-987.
  14. [14] M. K. Sabullah, S. A. M. Khalidi, D. N. A.Wahid, S. A. Sani, R. Abdulla, A. A. M. Faik, J. A. Gansau, S. A. Ahmad, and M. Y. Shukor. “Assessment of Monopterus albus liver as a source of Cholinesterase for the detection of heavy metals”. In: Journal of Physics: Conference Series. 1358. 1. IOP Publishing, 2019, 12029. DOI: 10.1088/1742-6596/1358/1/012029.
  15. [15] N. Nordin, R. R. Cletus, M. K. Sabullah, S. Aishah, M. Khalidi, R. Abdulla, and S. A. Ahmad, (2020) “Cholinesterase from the Liver of Diodon hystrix for Detection of Metal Ions" Pertanika Journal of Science and Technology 28(Special Issue 2): 107–119. DOI: 10.47836/pjst.28.S2.09.
  16. [16] A. A. Green and W. L. Hughes, (1955) “Protein fractionation on the basis of solubility in aqueous solutions of salts and organic solvents" Methods in Enzymology 1(C): 67–90. DOI: 10.1016/0076-6879(55)01014-8.
  17. [17] G. L. Classics Ellman, K. D. Courtney, V. Andres, and R. M. Featherstone, (1961) “A new and rapid colorimetric determination of acetylcholinesterase activity" Biochem Pharmacol 7: 88–95. DOI: 10.1016/0006-2952(61)90145-9.
  18. [18] M. M. Bradford, (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. DOI: 10.1016/0003-2697(76)90527-3.
  19. [19] M. K. Sabullah and M. E. Khayat, (2015) “Assessment of inhibitive assay for insecticides using acetylcholinesterase from Puntius schwanenfeldii" Journal of Biochemistry, Microbiology and Biotechnology 3(2): 26–29.
  20. [20] J.-R. Gao and K. Y. Zhu, (2001) “An acetylcholinesterase purified from the greenbug (Schizaphis graminum) with some unique enzymological and pharmacological characteristics" Insect Biochemistry and Molecular Biology 31(11): 1095–1104. DOI: 10.1016/S0965-1748(01)00057-1.
  21. [21] J. B. Salles, V. L. F. C. Bastos, M. V. Silva Filho, O. L. T. Machado, C. M. C. Salles, S. G. de Simone, and J. C. Bastos, (2006) “A novel butyrylcholinesterase from serum of Leporinus macrocephalus, a Neotropical fish" Biochimie 88(1): 59–68. DOI: 10.1016/j.biochi.2005.06.017.
  22. [22] H. Bisswanger, (2014) “Enzyme assays" Perspectives in Science 1(1-6): 41–55. DOI: 10.1016/j.pisc.2014.02.005.
  23. [23] N. V. Efremova, S. R. Sheth, and D. E. Leckband, (2001) “Protein-induced changes in poly (ethylene glycol) brushes: molecular weight and temperature dependence" Langmuir 17(24): 7628–7636. DOI: 10.1021/la010405c.
  24. [24] C. Mayberry, P. Mawson, and S. K. Maloney, (2015) “Plasma cholinesterase activity of rats, western grey kangaroos, alpacas, sheep, cattle, and horses" Journal of pharmacological and toxicological methods 72: 26–28. DOI: 10.1016/j.vascn.2015.01.003.
  25. [25] B. Nunes, (2011) “The use of cholinesterases in ecotoxicology" Reviews of environmental contamination and toxicology 212: 29–59. DOI: 10.1007/978-1-4419-8453-1_2.
  26. [26] M. K. Sabullah and M. E. Khayat, (2015) “Assessment of inhibitive assay for insecticides using acetylcholinesterase from Puntius schwanenfeldii" Journal of Biochemistry, Microbiology and Biotechnology 3(2): 26–29.
  27. [27] S. A. Ahmad, M. K. Sabullah, N. A. Shamaan, M. Y. Abd Shukor, H. Jirangon, A. Khalid, and M. A. Syed, (2016) “Evaluation of acetylcholinesterase source from fish, for detection of carbamate" Journal of environmental biology 37(4): 479. DOI: 27498490.
  28. [28] L. G. Tham, M. Y. Shukor, M. A. Syed, N. A. Shamaan, and A. R. Othman, (2017) “Partial Purification of Cholinesterase from Pangasius pangasius using Affinity Chromatography" Journal of Environmental Microbiology and Toxicology 5(2): 14–18.
  29. [29] S. A. M. Khalidi, M. K. Sabullah, S. A. Sani, S. A. Ahmad, M. Y. Shukor, I. N. M. Jaafar, and B. Gunasekaran. “Acetylcholinesterase from the brain of Monopterus albus as detection of metal ions”. In: Journal of Physics: Conference Series. 1358. 1. IOP Publishing, 2019, 12028. DOI: 10.1088/1742-6596/1358/1/012028.
  30. [30] S. L. M. Masson P and B. C. F, (2002) “Substrate activation in acetylcholinesterase induced by low pH or mutation in the -cation subsite" Biochemistry and Biophysics 1594: 313–324. DOI: 10.1016/S0167-4838(01)00323-5.
  31. [31] B. J. K. Fairbrother A, Marden B T and H. M. J, (1991) “Cholinesterase-inhibiting insecticides: Their impact on wildlife and the environment"Wildl Environ: 35–71.
  32. [32] L. Sawle and K. Ghosh, (2011) “How do thermophilic proteins and proteomes withstand high temperature?" Biophysical journal 101(1): 217–227. DOI: 10.1016/j.bpj.2011.05.059.
  33. [33] I. B. Wilson and E. Cabib, (1956) “Acetylcholinesterase: enthalpies and entropies of activation1" Journal of the American Chemical Society 78(1): 202–207. DOI: 10.1021/ja01582a056.
  34. [34] P. B. Armentrout, B. Yang, and M. T. Rodgers, (2013) “Metal cation dependence of interactions with amino acids: bond energies of Rb+ and Cs+ to Met, Phe, Tyr, and Trp" The Journal of Physical Chemistry B 117(14): 3771–3781. DOI: 10.1021/jp401366g.
  35. [35] J. Glusker, A. K. Katz, and C. Bock, (1999) “Metal ions in biological systems" The Rigaku Journal 16(2): 8–19. DOI: 10.1111/j.1469-185X.1953.tb01384.x.
  36. [36] D. P. Giedroc and A. I. Arunkumar, (2007) “Metal sensor proteins: nature’s metalloregulated allosteric switches" Dalton Transactions (29): 3107–3120. DOI: 10.1039/B706769K.
  37. [37] C. U. Eccles and Z. Annau, (1982) “Prenatal methyl mercury exposure: I. Alterations in neonatal activity" Neurobehavioral toxicology and teratology 4(3): 371–376.
  38. [38] T. W. Clarkson, G. F. Nordberg, and P. R. Sager, (1985) “Reproductive and developmental toxicity of metals" Scandinavian journal of work, environment health: 145–154. DOI: 10.5271/sjweh.2239.
  39. [39] A. Naji, F. R. Khan, and S. H. Hashemi, (2016) “Potential human health risk assessment of trace metals via the consumption of marine fish in Persian Gulf" Marine Pollution Bulletin 109(1): 667–671. DOI: 10.1016/j.marpolbul.2016.05.002.
  40. [40] A. Arulkumar, S. Paramasivam, and R. Rajaram, (2017) “Toxic heavy metals in commercially important food fishes collected from Palk Bay, Southeastern India" Marine Pollution Bulletin 119(1): 454–459. DOI: 10.1016/j.marpolbul.2017.03.045.
  41. [41] M. F. Frasco, J.-P. Colletier, M. Weik, F. Carvalho, L. Guilhermino, J. Stojan, and D. Fournier, (2007) “Mechanisms of cholinesterase inhibition by inorganic mercury" the FEBS Journal 274(7): 1849–1861. DOI: 10.1111/j.1742-4658.2007.05732.x.
  42. [42] M. S. Aidil, M. K. Sabullah, M. I. E. Halmi, R. Sulaiman, M. S. Shukor, M. Y. Shukor, N. A. Shaharuddin, M. A. Syed, and A. Syahir, (2013) “Assay for heavy metals using an inhibitive assay based on the acetylcholinesterase from Pangasius hypophthalmus (Sauvage, 1878)" Fresenius Environ Bull 22(12): 3572–3576. DOI: 10.1007/s12210-014-0359-0.
  43. [43] U. A. Muhammad, L. G. Tham, N. Perumal, H. M. Daud, N. A. Yasid, and M. Y. Shukor, (2016) “Assessment of acetylcholinesterase (AChE) from Oreochromis mossambicus (Cuvier, 1831) as a source of enzyme for insecticides detection" Bioremediation Science and Technology Research 4(2): 11–17.
  44. [44] A. F. Zulkifli, L. G. Tham, N. Perumal, M. K. Sabullah, A. M. Azzeme, M. Y. Shukor, and N. A. Shaharuddin, (2017) “Assay for heavy metals using an inhibitive assay based on the acetylcholinesterase from Channa striatus" Bioremediation Science and Technology Research 5(1): 7–11.


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