Isolation and screening of extracellular anticancer enzymes from halophilic and halotolerant bacteria from different saline environments in Iran

  • Mahdis Zolfaghar
  • Mohammad Ali AmoozegarEmail author
  • Khosro Khajeh
  • Hamid Babavalian
  • Hamid Tebyanian
Original Article


It was confirmed that several enzymes have anti-cancer activity. The enzymes l-asparaginase, l-glutaminase, and l-arginase were chosen according to amino acids starvation in cancer cells and screened in halophilic and halotolerant bacteria, given probably less immunological reactions of halophilic or halotolerant enzymes in patients. Out of 110 halophilic and halotolerant strains, isolated from different saline environments in Iran and screened, some could produce a variety of anticancer enzymes. A total of 29, 4, and 2 strains produced l-asparaginase, l-glutaminase, and l-arginase, respectively. According to the phenotypic characteristics and partial 16S rRNA gene sequence analysis, the positive strains—strains with the ability to produce these anticancer enzymes—were identified as the members of the genera: Bacillus, Dietzia, Halobacillus, Rhodococcus, Paenibacillus and Planococcus as Gram-positive bacteria and Pseudomonas, Marinobacter, Halomonas, Idiomarina, Vibrio and Stappia as Gram-negative bacteria. The production of anticancer enzymes was mostly observed in the rod-shaped Gram-negative isolates, particularly in the members of the genera Halomonas and Marinobacter. Most of the enzymes were produced in the stationary phase of growth and the maximum enzyme activity was experienced in strain GBPx3 (Vibrio sp.) for l-asparaginase at 1.0 IU/ml, strain R2S25 (Rhodococcus sp.) for l-glutaminase at 0.6 IU/ml and strain GAAy3 (Planococcus sp.) for l-arginase at 3.1 IU/ml. The optimum temperature and pH for l-asparaginase and l-glutaminase activities in selected strains were similar to the physiological conditions of human body and the enzymes could tolerate NaCl up to 7.5% concentration.


Anticancer enzymes Halophiles l-asparaginase Halophilic Halotolerant enzymes 



This work was supported by a Grant from the Research Council of University of Tehran.

Compliance with ethical standards

Conflict of interest

There is no conflict of interest.

Supplementary material

11033_2019_4787_MOESM1_ESM.docx (24 kb)
Supplementary material 1 (DOCX 23 kb)
11033_2019_4787_MOESM2_ESM.jpg (76 kb)
Supplementary material 2 (JPEG 76 kb)
11033_2019_4787_MOESM3_ESM.jpg (54 kb)
Supplementary material 3 (JPEG 53 kb)


  1. 1.
    Lodish HF, Berk A, Zipursky SL, Matsudaira P, Baltimore D, Darnell J (2000) Molecular cell biology, vol 4. WH Freeman, New YorkGoogle Scholar
  2. 2.
    Ciardiello F, Tortora G (2001) A novel approach in the treatment of cancer: targeting the epidermal growth factor receptor. Clin Cancer Res 7(10):2958–2970Google Scholar
  3. 3.
    Gonzalez N (1969) Enzyme therapy and cancer. Metabolic PathwaysGoogle Scholar
  4. 4.
    Wheatley DN (2004) Controlling cancer by restricting arginine availability-arginine-catabolizing enzymes as anticancer agents. Anticancer Drugs 15(9):825–833Google Scholar
  5. 5.
    Cantor JR, Panayiotou V, Agnello G, Georgiou G, Stone EM (2012) Engineering reduced-immunogenicity enzymes for amino acid depletion therapy in cancer. Methods Enzymol 502:291–319Google Scholar
  6. 6.
    Kreis W, Hession C (1973) Biological effects of enzymatic deprivation of l-methionine in cell culture and an experimental tumor. Cancer Res 33(8):1866–1869Google Scholar
  7. 7.
    Agrawal V, Alpini SE, Stone EM, Frenkel EP, Frankel AE (2012) Targeting methionine auxotrophy in cancer: discovery and exploration. Expert Opin Biol Ther 12(1):53–61Google Scholar
  8. 8.
    Phillips MM, Sheaff MT, Szlosarek PW (2013) Targeting arginine-dependent cancers with arginine-degrading enzymes: opportunities and challenges. Cancer Res Treat 45(4):251–262Google Scholar
  9. 9.
    Niemeyer CM, Hitchcock-Bryan S, Sallan SE (1985) Comparative analysis of treatment programs for childhood acute lymphoblastic leukemia. Semin Oncol 12(2):122–130Google Scholar
  10. 10.
    Greenberg DM, Blumenthal G, Ramadan M-EA (1964) Effect of administration of the enzyme glutaminase on the growth of cancer cells. Cancer Res 24(6):957–963Google Scholar
  11. 11.
    Scott L, Lamb J, Smith S, Wheatley D (2000) Single amino acid (arginine) deprivation: rapid and selective death of cultured transformed and malignant cells. Br J Cancer 83(6):800Google Scholar
  12. 12.
    Kallio R, Larson A (1955) Methionine degradation by a species of Pseudomonas. In: McElroy WD, Glass B (eds) A symposium on amino acid metabolism. Johns Hopkins Press, pp 616–631Google Scholar
  13. 13.
    Broome J (1961) Evidence that the l-asparaginase activity of guinea pig serum is responsible for its antilymphoma effects. Nature 191(4793):1114Google Scholar
  14. 14.
    Cooney D, Handschumacher R (1970) l-asparaginase and l-asparagine metabolism. Annu Rev Pharmacol 10(1):421–440Google Scholar
  15. 15.
    El-Sayed AS (2010) Microbial l-methioninase: production, molecular characterization, and therapeutic applications. Appl Microbiol Biotechnol 86(2):445–467Google Scholar
  16. 16.
    Feun L, Savaraj N (2006) Pegylated arginine deiminase: a novel anticancer enzyme agent. Expert Opin Investig Drugs 15(7):815–822Google Scholar
  17. 17.
    Zarparvar P, Amoozegar MA, Babavalian H, Reza FM, Tebyanian H, Shakeri F (2016) Isolation and identification of culturable halophilic bacteria with producing hydrolytic enzyme from Incheh Borun Hypersaline Wetland in Iran. Cell Mol Biol 62(12):31–36. Google Scholar
  18. 18.
    Taherian A, Fazilati M, Moghadam AT, Tebyanian H (2018) Optimization of purification procedure for horse F(abʹ)2 antivenom against Androctonus crassicauda (Scorpion) venom. Trop J Pharm Res 17(3):409–414. Google Scholar
  19. 19.
    Khomarlou N, Aberoomand-Azar P, Lashgari AP, Tebyanian H, Hakakian A, Ranjbar R, Ayatollahi SA (2018) Essential oil composition and in vitro antibacterial activity of Chenopodium album subsp. striatum. Acta Biol Hung 69(2):144–155. Google Scholar
  20. 20.
    Fukuchi S, Yoshimune K, Wakayama M, Moriguchi M, Nishikawa K (2003) Unique amino acid composition of proteins in halophilic bacteria. J Mol Biol 327(2):347–357Google Scholar
  21. 21.
    Kamekura M (1986) Production and function of enzymes of eubacterial halophiles. FEMS Microbiol Ecol 2(1–2):145–150Google Scholar
  22. 22.
    Galinski E, Tindall B, Herbert R, Sharp R (1992) Biotechnological prospects for halophiles and halotolerant micro-organisms. In: Molecular biology and biotechnology of extremophiles. Blackie Publishing Group, pp 76–114Google Scholar
  23. 23.
    Rezaeeyan Z, Safarpour A, Amoozegar MA, Babavalian H, Tebyanian H, Shakeri F (2017) High carotenoid production by a halotolerant bacterium, Kocuria sp. strain QWT-12 and anticancer activity of its carotenoid. EXCLI J 16:840–851. Google Scholar
  24. 24.
    Ebrahiminezhad A, Rasoul-Amini S, Ghasemi Y (2011) l-asparaginase production by moderate halophilic bacteria isolated from Maharloo Salt Lake. Indian J Microbiol 51(3):307–311Google Scholar
  25. 25.
    Sivakumar K, Sahu MK, Manivel P, Kannan L (2006) Optimum conditions for l-glutaminase production by actinomycete strain isolated from estuarine fish, Chanos chanos (Forskal, 1775). Indian J Exp Biol 44(3):256Google Scholar
  26. 26.
    Murray R, Doetsch R, Robinow C (1994) Methods for general and molecular bacteriology. Determination and cytological light microscopy. American Society for Microbiology, Washington, DC, pp 31–32Google Scholar
  27. 27.
    Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4(4):406–425Google Scholar
  28. 28.
    Babavalian H, Latifi AM, Shokrgozar MA, Bonakdar S, Tebyanian H, Shakeri F (2016) Cloning and expression of recombinant human platelet-derived growth factor-BB in Pichia Pink. Cell Mol Biol 62(8):45–51. Google Scholar
  29. 29.
    Tebyanian H, Mirhosseiny SH, Kheirkhah B, Hassanshahian M (2014) Isolation and identification of Mycoplasma synoviae from suspected ostriches by polymerase chain reaction, in Kerman Province, Iran. Jundishapur J Microbiol 7(9):e19262Google Scholar
  30. 30.
    Gulati R, Saxena R, Gupta R (1997) A rapid plate assay for screening l-asparaginase producing micro-organisms. Lett Appl Microbiol 24(1):23–26Google Scholar
  31. 31.
    Sundar WA, Nellaiah H (2013) A rapid method for screening of methioninase producing Serratia marcescens species from soil. Int J Pharm Pharm Sci 5(2):426–427Google Scholar
  32. 32.
    Mendz GL, Holmes EM, Ferrero RL (1998) In situ characterization of Helicobacter pylori arginase. Biochim Biophys Acta Protein Struct Mol Enzymol 1388(2):465–477Google Scholar
  33. 33.
    Hirsch-Kolb H, Heine JP, Kolb HJ, Greenberg DM (1970) Comparative physical–chemical studies of mammalian arginases. Comp Biochem Physiol 37(3):345–359Google Scholar
  34. 34.
    McGee DJ, Zabaleta J, Viator RJ, Testerman TL, Ochoa AC, Mendz GL (2004) Purification and characterization of Helicobacter pylori arginase, RocF: unique features among the arginase superfamily. Eur J Biochem 271(10):1952–1962Google Scholar
  35. 35.
    Mia AS, Koger HD (1978) Direct colorimetric determination of serum arginase in various domestic animals. Am J Vet Res 39(8):1381–1383Google Scholar
  36. 36.
    Wade H, Robinson H, Phillips B (1971) Asparaginase and glutaminase activities of bacteria. J Gen Microbiol 69(3):299–312Google Scholar
  37. 37.
    Amer MN, Mansour NM, El-Diwany AI, Dawoud IE, Rashad FM (2013) Isolation of probiotic lactobacilli strains harboring l-asparaginase and arginine deiminase genes from human infant feces for their potential application in cancer prevention. Ann Microbiol 63(3):1121–1129Google Scholar
  38. 38.
    Rohban R, Amoozegar MA, Ventosa A (2009) Screening and isolation of halophilic bacteria producing extracellular hydrolyses from Howz Soltan Lake, Iran. J Ind Microbiol Biotechnol 36(3):333–340Google Scholar
  39. 39.
    Zarparvar P, Amoozegar MA, Nikou MM, Schumann P, Ventosa A (2014) Salinithrix halophila gen. nov., sp. nov., a halophilic bacterium in the family Thermoactinomycetaceae. Int J Syst Evol Microbiol 64(12):4115–4119. Google Scholar
  40. 40.
    Mehrshad M, Amoozegar MA, Didari M, Bagheri M, Fazeli SAS, Schumann P, Spröer C, Sánchez-Porro C, Ventosa A (2013) Bacillus halosaccharovorans sp. nov., a moderately halophilic bacterium from a hypersaline lake. Int J Syst Evol Microbiol 63(8):2776–2781. Google Scholar
  41. 41.
    Hassanshahian M, Mohamadian J (2011) Isolation and characterization of Halobacterium salinarum from saline lakes in Iran. Jundishapur J Microbiol 4(5):59–66Google Scholar
  42. 42.
    Azadian F, Badoei-dalfard A, Namaki-Shoushtari A, Karami Z, Hassanshahian M (2017) Production and characterization of an acido-thermophilic, organic solvent stable cellulase from Bacillus sonorensis HSC7 by conversion of lignocellulosic wastes. J Genet Eng Biotechnol 15(1):187–196Google Scholar
  43. 43.
    Mahajan RV, Saran S, Kameswaran K, Kumar V, Saxena R (2012) Efficient production of l-asparaginase from Bacillus licheniformis with low-glutaminase activity: optimization, scale up and acrylamide degradation studies. Bioresour Technol 125:11–16Google Scholar
  44. 44.
    Kafkewitz D, Goodman D (1974) l-asparaginase production by the rumen anaerobe Vibrio succinogenes. J Appl Microbiol 27(1):206–209Google Scholar
  45. 45.
    Kumar SA, Arunasri R, Jayachandra Y, Sulochana M (2010) Screening of extra screening of extracellular hydrolytic enzymes from Marinobacter hydrocarbonoclasticus strain ak5. Bioscan 5(1):97–99Google Scholar
  46. 46.
    Manna S, Sinha A, Sadhukhan R, Chakrabarty S (1995) Purification, characterization and antitumor activity of l-asparaginase isolated from Pseudomonas stutzeri MB-405. Curr Microbiol 30(5):291–298Google Scholar
  47. 47.
    Cook WR, Hoffman JH, Bernlohr RW (1981) Occurrence of an inducible glutaminase in Bacillus licheniformis. J Bacteriol 148(1):365Google Scholar
  48. 48.
    Biswas J, Paul A (2013) Production of extracellular enzymes by halophilic bacteria isolated from solar salterns. IJABPT 4(4):30–36Google Scholar
  49. 49.
    Shirazian P, Asad S, Amoozegar MA (2016) The potential of halophilic and halotolerant bacteria for the production of antineoplastic enzymes: l-asparaginase and l-glutaminase. EXCLI J 15:268Google Scholar
  50. 50.
    Yu J-J, Park K-B, Kim S-G, Oh S-H (2013) Expression, purification, and biochemical properties of arginase from Bacillus subtilis 168. J Microbiol 51(2):222–228Google Scholar
  51. 51.
    Ventosa A (1988) Taxonomy of moderately halophilic heterotrophic eubacteria. Halophilic Bact 1:71–84Google Scholar
  52. 52.
    Albanese E, Kafkewitz K (1978) Effect of medium composition on the growth and asparaginase production of Vibrio succinogenes. Appl Environ Microbiol 36(1):25–30Google Scholar
  53. 53.
    Abbas MA, Askar SN (2014) Production, purification and characterization of extracellular l-asparaginase from Erwinia carotovora. Int J Bioassays 3(12):3553–3559Google Scholar
  54. 54.
    Sathish T, Prakasham RS (2010) Enrichment of glutaminase production by Bacillus subtilis RSP-GLU in submerged cultivation based on neural network—genetic algorithm approach. J Chem Technol Biotechnol 85(1):50–58Google Scholar
  55. 55.
    Nagendra Prabhu G, Chandrasekaran M (1997) Impact of process parameters on l-glutaminase production by marine Vibrio costicola in solid state fermentation using polystyrene as an inert support. Process Biochem 32(4):285–289Google Scholar
  56. 56.
    Jennings MP, Beacham IR (1990) Analysis of the Escherichia coli gene encoding l-asparaginase II, ansB, and its regulation by cyclic AMP receptor and FNR proteins. J Bacteriol 172(3):1491–1498Google Scholar
  57. 57.
    Distasio JA, Niederman RA, Kafkewitz D, Goodman D (1976) Purification and characterization of l-asparaginase with anti-lymphoma activity from Vibrio succinogenes. J Biol Chem 251(22):6929–6933Google Scholar
  58. 58.
    Li L-Z, Xie T-H, Li H-J, Qing C, Zhang G-M, Sun M-S (2007) Enhancing the thermostability of Escherichia coli l-asparaginase II by substitution with pro in predicted hydrogen-bonded turn structures. Enzyme Microb Technol 41(4):523–527Google Scholar
  59. 59.
    Singh P, Banik R (2013) Biochemical characterization and antitumor study of l-glutaminase from Bacillus cereus MTCC 1305. Appl Biochem Biotechnol 171(2):522–531Google Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.Extremophiles Laboratory, Department of Microbiology, School of Biology and Center of Excellence in Phylogeny of Living Organisms, College of ScienceUniversity of TehranTehranIran
  2. 2.Department of Biochemistry, Faculty of Biological ScienceTarbiat Modares UniversityTehranIran
  3. 3.Applied Virology Research CenterBaqiyatallah University of Medical SciencesTehranIran
  4. 4.Research Center for Prevention of Oral and Dental DiseasesBaqiyatallah University of Medical SciencesTehranIran

Personalised recommendations