Enzymatic cyanide detoxification by partially purified cyanide dihydratase obtained from Serratia marcescens strain AQ07.

  • K. I. Karamba
  • M. K. Sabullah
  • A. Zulkharnain
  • C. Gomez-Fuentes
  • S. A. Ahmad
Keywords: Serratia marcescens, detoxification, enzyme, cyanide dihydratase


The partially purified enzyme of indigenously isolated Serratia marcescensstrain AQ07 was utilised to develop the best form of cyanide detoxification method that is eco-friendly and cost effective. The present study evaluates the feasibility of the enzyme to degrade high cyanide concentrations and the possible metabolic pathways involved, for which the protein concentration and cyanide detoxification activity were quantified. Bacterial cells grown in cyanide incorporated medium were disrupted by sonication and the resultant cell free extract were tested for metabolic pathway. The cell free extract was precipitated by ammonium sulphate precipitation and partially purified by ion exchange chromatography using DEAE cellulose. The maximum enzyme activity achieved was 2125 µM/min. The partially purified enzyme was found to be able to detoxify 82% of 2 mM KCN in 10 min of incubation and cyanide degradation (or depletion) rate showing a linear increase with increasing enzyme concentration. The effective accruing of ammonia as metabolite illustrated that the detoxification was ensued via the function of cyanide dihydratase. Additional confirmation through SDS-Page showed that the molecular weight of enzyme was assessed to be ~38 kDa, which is tandem with the reported cyanide dihydratases. Hence, the use of enzyme as a substitute to live bacterial cells in detoxification of cyanide illustrates various advantages such as the capacity to withstand and detoxify higher cyanide concentration and total reduction in the total cost of process since nutrient provision is immaterial.


Adams, D.J., Komen, J.V. and Pickett, T.M. (2001). Biological cyanide degradation. In: Cyanide: Social, Industrial and Econmic Aspects, (C. Young, ed.), Pp. 203–213. The Metals Society, Warrendale.

Ahmad, S.A., Shamaan, N.A., Syed, M.A., Khalid, A., Ab Rahman, N.A., Abdul Khalil, K., Dahalan, F.A. and Shukor, M.Y. (2017). Meta-cleavage pathway of phenol degradation by Acinetobacter sp. strain AQ5NOL 1. Rendiconti Lincei 28, 1-9.

Akcil, A., Karahan, A.G., Ciftci, H. and Sagdic, O. (2003). Biological treatment of cyanide by natural isolated bacteria (Pseudomonas sp.). Minerals Engineering 16, 643-649.

Anderson, P.M. (1980). Purification and properties of the inducible enzyme cyanase. Biochemistry (Mosc.) 19, 2882-2888.

Balagurusamy, N. (2005). Anaerobic bioremediation–an untapped potential. Revista Mexicana De Ingenieria Quimica 4, 273-287.

Basheer, S., Kut, Ö., Prenosil, J.E. and Bourne, J,R. (1992). Kinetics of enzymatic degradation of cyanide. Biotechnology and Bioengineering 39, 629-634.

Bertrand, B., Martínez-Morales, F. and Trejo-Hernández, M.R. (2013). Fungal laccases: induction and production. Revista Mexicana de Ingeniería Química 12, 473-488.

Braun, V., Rehn, K. and Wolff, H. (1970). Supramolecular structure of the rigid layer of the cell wall of Salmonella, Serratia, Proteus, and Pseudomonas fluorescens. Number of lipoprotein molecules in a membrane layer. Biochemistry (Mosc.) 9, 5041-5049.

Dash, R.R., Gaur, A. and Balomajumder, C. (2009). Cyanide in industrial wastewaters and its removal: a review on biotreatment. Journal of Hazardous Materials 163, 1-11.

Dorr, P.K. and Knowles, C.J. (1989). Cyanide oxygenase and cyanase activities of Pseudomonas fluorescens NCIMB 11764. FEMS Microbiology Letters 60, 289-294.

Dursun, A.Y. and Aksu, Z. (2000). Biodegradation kinetics of ferrous (II) cyanide complex ions by immobilized Pseudomonas fluorescens in a packed bed column reactor. Process Biochemistry 35, 615-622.

Ebbs, S. (2004). Biological degradation of cyanide compounds. Current Opinion in Biotechnology 15, 231-236.

Ebbs, S.D., Kosma, D.K., Nielson, E.H., Machingura, M., Baker, A.J. and Woodrow, I.E. (2010). Nitrogen supply and cyanide concentration influence the enrichment of nitrogen from cyanide in wheat (Triticum aestivum L.) and sorghum (Sorghum bicolor L.). Plant, Cell and Environment 33, 1152-1160.

Esquivel-Viveros, A., Ponce-Vargas, F., Esponda-Aguilar, P., Prado-Barragán, L.A., Gutiérrez-Rojas, M., Lye G.J. and Huerta-Ochoa. S. (2009) Biodegradation of [bmim][PF6] using Fusarium sp. Revista Mexicana de Ingeniería Química 8, 163-168.

Halmi, M.I.E., Wasoh, H., Sukor, S., Ahmad, S.A., Yusof, M.T. and Shukor, M.Y. (2014). Bioremoval of molybdenum from aqueous solution. International Journal of Agriculture and Biology 16, 848-850.

Harris, R.E. and Knowles, C.J. (1983). The conversion of cyanide to ammonia by extracts of a strain of Pseudomonas fluorescens that utilizes cyanide as a source of nitrogen for growth. FEMS Microbiology Letters 20, 337-341.

Ingvorsen, K., Højer-Pedersen, B. and Godtfredsen, S.E. (1991). Novel cyanide-hydrolyzing enzyme from Alcaligenes xylosoxidans subsp. denitrificans. Applied and Environmental Microbiology 57, 1783-1789.

Interiano-López, M., Ramírez-Coutiño, V., Godinez-Tovar, L., Zamudio-Pérez, E. and Rodríguez-Valadez, F. (2019). Bioremediation methods assisted with humic acid for the treatment of oil-contaminated drill cuttings. Revista Mexicana De Ingeniería Química 18, 929-937.

Jandhyala, D., Berman, M., Meyers, P.R., Sewell, B.T., Willson, R.C. and Benedik, M.J. (2003). CynD, the cyanide dihydratase from Bacillus pumilus: Gene cloning and structural studies. Applied of Environmental Microbiology 69, 4794-4805.

Karamba, K.I., Ahmad, S.A., Zulkharnain, A., Yasid, N.A., Ibrahim, S. and Shukor, M.Y. (2018). Batch growth kinetic studies of locally isolated cyanide-degrading Serratia marcescens strain AQ07. 3 Biotech (2018) 8: 11. https://doi.org/10.1007/s13205-017-1025-x

Karamba, K.I., Ahmad, S.A., Zulkharnain, A., Syed, M.A., Abdul Khalil, K., Shamaan, N.A., Dahalan, F.A. and Shukor, M.Y. (2016). Optimisation of biodegradation conditions for cyanide removal by Serratia marcescens strain AQ07 using one-factor-at-a-time technique and response surface methodology. Rendiconti Lincei 27, 533-545.

Karamba, K.I., Ahmad, S.A., Zulkharnain, A., Yasid, N.A., Khalid, A. and Shukor, M.Y. (2017). Biodegradation of cyanide and evaluation of kinetic models by immobilized cells of Serratia marcescens strain AQ07. International Journal of Environmental Science and Technology 14, 1945-1958.

Karamba, K.I., Shukor, M.Y., Syed, M.A., Zulkharnain, A., Yasid, N.A., Khalid, A., Abdul Khalil, K. and Ahmad, S.A. (2015a). Isolation, screening and characterisation of cyanide-degrading Serratia marcescens strain AQ07. Journal of Chemical and Pharmaceutical Sciences 8, 401-406.

Karamba, K., Syed, M.A., Shukor, M.Y.. and Ahmad, S.A. (2014) Effect of heavy metals on cyanide biodegradation by resting cells of Serratia marcescens strain AQ07. Journal of Environmental Microbiology and Toxicology 2, 17–20.

Karamba, K.I., Syed, M.A., Shukor, M.Y. and Ahmad, S.A. (2015b). Biological remediation of cyanide: A Review. Biotropia 22, 151-163.

Kunz, D.A., Chen, J.L. and Pan, G. (1998). Accumulation of α-keto acids as essential components in cyanide assimilation by Pseudomonas fluorescens NCIMB 11764. Applied and Environmental Microbiology 64, 4452-4459.

Kunz, D.A., Nagappan, O., Silva-Avalos, J. and Delong, G.T. (1992). Utilization of cyanide as nitrogenous substrate by Pseudomonas fluorescens NCIMB 11764: Evidence for multiple pathways of metabolic conversion. Applied and Environmental Microbiology 58, 2022-2029.

Kunz, D.A., Wang, C.S. and Chen, J.L. (1994). Alternative routes of enzymic cyanide metabolism in Pseudomonas fluorescens NCIMB 11764. Microbiology 140, 1705-1712.

Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685.

Lowry, C.O., Rosebrough, N., Farr, A. and Randall, R.J. (1951). Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry 193, 265-275.

Maniyam, M.N., Sjahrir, F., Ibrahim, A.L., Ibrahim, A.L. and Cass, A.E. (2013). Biodegradation of cyanide by acetonitrile-induced cells of Rhodococcus sp. UKMP-5M. The Journal of General and Applied Microbiology 404, 393-404.

Maniyam, N.M., Sjahrir, F., Ibrahim, A.L. and Cass, A.E. (2015). Enzymatic cyanide degradation by cell-free extract of Rhodococcus UKMP- 5M. Journal of Environmental Science and Health, Part A 50, 357-364.

Martínez-Sánchez, J., Membrillo-Venegas, I., Martínez-Trujillo, A. and García-Rivero, A. (2018). Decolorization of reactive black 5 by immobilized Trametes versicolor. Revista Mexicana De Ingeniería Química 17, 107-121.

Meyers, P.R., Gokool, P., Rawlings, D.E. and Woods, D.R. (1991). An efficient cyanide-degrading Bacillus pumilus strain. Journal of General Microbiology 137, 1397-1400.

Meyers, P.R., Rawlings, D.E., Woods, D.R. and Lindsey, G.G. (1993). Isolation and characterization of a cyanide dihydratase from Bacillus pumilus Cl. Journal of Bacteriology 175, 6105-6112.

Nagashima, S. (1977). Spectrophotometric determination of cyanide with ɤ - picoline - barbituric acid. Analytica Chimica Acta A 91, 303-306.

Nolan, L.M., Harnedy, P.A., Turner, P., Hearne, A.B. and O'Reilly, C. (2003). The cyanide hydratase enzyme of Fusarium lateritium also has nitrilase activity. FEMS Microbiology Letters 221, 161–165.

Parmar, P., Soni, A. and Desai, P. (2013). Enzymatic study of cyanide utilizing Pseudomonas species isolated from contaminated soil. Journal of Scientific and Innovative Research 2, 1058-1066.

Perumal, M., Prabakaran, J. and Kmaraj, M. (2013). Isolation and characterization of potential cyanide degrading Bacillus nealsonii from different industrial effluents. International Journal of ChemTech Research 5, 2357-2364.

Potivichayanon, S. and Kitleartpornpairoat, R. (2010). Biodegradation of cyanide by a novel cyanide-degrading bacterium. World Academy of Science, Engineering and Technology 42, 1362-1365.

Rand, M.C., Greenberg, A.E. and Taras, M.J. (1976). Standard methods for the examination of water and wastewater. Prepared and published jointly by American Public Health Association, American Water Works Association, and Water Pollution Control Federation.

Roberts, R.L. and Cabib, E. (1982). Serratia marcescens chitinase: one-step purification and use for the determination of chitin. Anal Biochemistry 27, 402-412.

Sabullah, M.K., Rahma, M.F., Ahmad, S.A., Sulaiman, M.R., Shukor, M.S., Gansau, A.J., Shamaan, N.A. and
Shukor, M.Y. (2017). Isolation and characterization of a molybdenum-reducing and phenolic- and catechol-degrading Enterobacter sp. strain SAW-2. Biotropia 24, 47-58.

Scopes, R.K. (2013) Protein purification: principles and practice. Springer, New York. https://doi.org/10.1007/978-1-4757-2333-5.

Watanabe, A., Yano, K., Kazuyoshi, Y. and Karube, I. (1998). Cyanide hydrolysis in a cyanide-degrading bacterium, Pseudornonas stutzeri AK61, by cyanidase. Microbiology 144, 1677-1682.

White, J.M., Jones, D.D., Huang, D. and Gauthier, J.J. (1988). Conversion of cyanide to formate and ammonia by a Pseudomonad obtained from industrial wastewater. Journal of Industrial Microbiology 3, 263-272.

Whitlock, J.L. and Mudder, T.I. (1986). The Homestake Wastewater Treatment Process: Biological Removal of Toxic Parameters From Cyanidation Wastewaters and Bioassay Effluent Evaluation. Elsevier, Amsterdam.
How to Cite
Karamba, K., Sabullah, M., Zulkharnain, A., Gomez-Fuentes, C., & Ahmad, S. (2019). Enzymatic cyanide detoxification by partially purified cyanide dihydratase obtained from Serratia marcescens strain AQ07. Revista Mexicana De Ingeniería Química, 19(2), 717-730. https://doi.org/10.24275/rmiq/Bio785

Most read articles by the same author(s)