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Volume: 6, Issue: 5, Sep-Oct, 2018
DOI: 10.7324/JABB.2018.60503

Research Article

Submerged fermentation, purification, and characterization of L-asparaginase from Streptomyces sp. isolated from soil

Savitha S. Desai, Basavaraj S. Hungund

  Author Affiliations


Actinomycetes are most valuable microbes because they are reported to produce different kinds of novel antibiotics and bioactive compounds. L-asparaginase enzyme is a chemotherapeutic agent used in the treatment of cancer. The present study reports production and characterization of purified L-asparaginase from an actinomycetes isolate. The isolate (A-164) was found to be a potent producer of L-asparaginase with an enzyme activity of 26.67 international unit (IU)/ml. Biochemical characterization of the isolate indicated that it belongs to genus Streptomyces. Laboratory scale enzyme production studies were undertaken using the standard medium. Further, the enzyme was purified by salt precipitation, anion exchange chromatography, and size exclusion chromatography. The purified enzyme exhibited the specific activity of 390 IU/mg with 60-fold purification and yield of 42% with a molecular weight of 34 KDa. The purified enzyme demonstrated the highest activity at pH 6.0 and temperature 35°C. The enzyme retained its activity till temperature of 50°C, indicating that it is a thermostable enzyme. A study on the effect of various additives on enzyme activity was also undertaken. The Km and Vmax values for the purified L-asparaginase were 0.065 mM and 20.80 IU/ml, respectively. The enzyme can further be used for health care and industrial applications.


L-asparaginase, Streptomyces sp., Biochemical characterization, Chromatography, Sodium dodecyl sulfatepolyacrylamide gel electrophoresis, Kinetic studies.

Citation: Desai SS, Hungund BS. Submerged fermentation, purification, and characterization of L-asparaginase from Streptomyces sp. isolated from soil. J App Biol Biotech. 2018;6(05):17-23. http://doi.org/10.7324/JABB.2018.60503.

Copyright: Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License, which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited.


1. Subramani R, Aalbersberg W. Marine actinomycetes: An ongoing source of novel bioactive metabolites. Microbiological Research. 2012; 167:571-580. https://doi.org/10.1016/j.micres.2012.06.005

2. Berdy J. Bioactive microbial metabolites. The Journal of Antibiotics. 2005; 58:1-26. https://doi.org/10.1038/ja.2005.1

3. Burge HM. Aerobiologist, EP Return to index. Clinical Microbiology Reviews. 1994; 7:357-417.

4. Desai SS, Chopra SJ, Hungund BS. Production, purification and characterization of L-Glutaminase from Streptomyces sp. isolated from soil. Journal of Applied Pharmaceutical Science. 2016; 6:100-105. https://doi.org/10.7324/JAPS.2016.60715

5. Piatkowska-Jakubas B, Krawczyk-Kulis M, Giebel S, Adamczyk-Cioch M, Czyz A, Lech-Maranda E, Paluszewska M, Palynyczko G, Piszcz J, Holowiecki J. Use of L-Asparaginase in acute lymphoblastic leukemia: recommendations of the Polish Adult Leukemia Group. Polish Archives of Internal Medicine. 2008; 118:664-669.

6. Sinha R, Singh HR, Jha SK. Microbial L-Asparaginase:Present and future prospective. International Journal for Innovative Research in Science & Technology. 2013; 2:7031-7051.

7. Asthana N, Azmi W. Microbial L-Asparaginase: A potent antitumour enzyme. Indian Journal of Biotechnology. 2003; 2:183-194.

8. Hanif N. Extraction of Anticancerous Enzymes from E.coli and a New Method to Study its Activity. Journal of Bacteriology & Parasitology. 2017; 8:1-10. https://doi.org/10.4172/2155-9597.1000301

9. McCredie KB, Ho DH, Freireich EJ. L‐Asparaginase for the treatment of cancer. CA: A Cancer Journal of Clinicians. 1973; 23:220-227. https://doi.org/10.3322/canjclin.23.4.220

10. El-Naggar NEA, El-Ewasy SM, El-Shweihy NM. Microbial L-Asparaginase as apotential therapeutic agent for treatment of Acute Lymphoblastic Leukemia:The Pros and Cons. International Journal of Pharmacy. 2014; 10:182-199. https://doi.org/10.3923/ijp.2014.182.199

11. Dejong PJ. L-Asparaginase production by Streptomyces griseus. Journal of Applied Microbiology. 1972; 23:1163-1164.

12. Kavitha A,Vijayalakshmi M. Optimization and Purification of L-Asparaginase Produced by Streptomyces tendae TK-VL_333. Zeitschrift f\ür Naturforschung C. 2010; 65:528-531. https://doi.org/10.1515/znc-2010-7-817

13. Sahu MK, Sivakumar K, Poorani E, Thangaradjou T, Kannan L. Studies on L-Asparaginase enzyme of actinomycetes isolated from estuarine fishes. Journal of Environmental Biology. 2007; 28:465-474.

14. Varma Ravi A, Sushma Kanapala, Naga Sai Babu V, Bodaiah B, Poda S. Partial Purification, Characterization and optimization of anti-leukemic enzyme L-Asparaginase from mangrove soil actinobacteria. Journal of Pharmacy Research. 2016; 10:502-511.

15. El-Sabbagh SM, El-Batanony NH, Salem TA. L-Asparaginase produced by Streptomyces strain isolated from Egyptian soil: Purification, characterization and evaluation of its anti-tumor. African Journal of Microbiology Research. 2013; 7:5677-5686. https://doi.org/10.5897/AJMR2013.1937

16. Kavitha A,Vijayalakshmi, M. A study on L-Asparaginase of Nocardia levis MK-VL_113. The Scientific World Journal. 2012; 1-5. https://doi.org/10.1100/2012/160434

17. Basha NS, Rekha R, Komala M, Ruby S. Production of extracellular anti-leukaemic enzyme L-Asparaginase from marine actinomycetes by solid state and submerged fermentation: Purification and characterization. Tropical Journal of Pharmaceutical Research. 2009; 8:353-360. https://doi.org/10.4314/tjpr.v8i4.45230

18. Mohamed SA, Elshal MF, Kumosani TA, Aldahlawi AM. Purification and characterization of Asparaginase from Phaseolus vulgaris seeds. Evidence-Based Complementary and Alternative Medicine. 2015; 1-6.

19. Bhagat J, Kaur A, Chadha BS. Single step purification of Asparaginase from endophytic bacteria Pseudomonas oryzihabitans exhibiting high potential to reduce acrylamide in processed potato chips. Food and Bioproducts Processing. 2016; 99:222-30. https://doi.org/10.1016/j.fbp.2016.05.010

20. Kiranmayi MU, Poda S, Vijayalakshmi M. Production and optimization of L-Asparaginase by an actinobacterium isolated from Nizampatnam mangrove ecosystem. Journal of Environmental Biology. 2014; 35:799-805.

21. Senthil Kumar M, Selvam K. Isolation and purification of high efficiency L-Asparaginase by quantitative preparative continuous-elution SDS PAGE electrophoresis. Journal of Microbial and Biochemical Technology. 2011; 3:73-83. https://doi.org/10.4172/1948-5948.1000055

22. Kamble VP, Rao RS, Borkar PS. Purification of L-Asparaginase from a bacteria Erwinia carotovora and effect of a dihydropyrimidine derivative on some of its kinetic parameters. Indian Journal of Biochemistry Biotechnology. 2006; 43:391-394.

23. El-Bessoumy AA, Sarhan M, Mansour J. Production, isolation, and purification of L-Asparaginase from Pseudomonas aeruginosa 50071 using solid-state fermentation. BMB Reports. 2004; 37:387-393. https://doi.org/10.5483/BMBRep.2004.37.4.387

24. Abdelghani T, Kurad BR, Poluri E. Antibacterial activity of bacterial isolates of soil bacteria collected from Palestine. Current Trends in Biotechnology and Pharmacy. 2009; 3:197-203.

25. Mahajan RV, Saran S, Saxena RK, Srivastava AK. A rapid, efficient and sensitive plate assay for detection and screening of L-Asparaginase-producing microorganisms. FEMS Microbiology Letters. 2013; 341:122-126. https://doi.org/10.1111/1574-6968.12100

26. Gulati R, Saxena RK, Gupta R. A rapid plate assay for screening L-Asparaginase producing micro‐organisms. Letters in Applied Microbiology. 1997; 24:23-26. https://doi.org/10.1046/j.1472-765X.1997.00331.x

27. Shirling EB, Gottlieb D. Methods for characterization of Streptomycetes species. International Journal of Systematic Bacteriology. 1966; 16:313-340. https://doi.org/10.1099/00207713-16-3-313

28. Peterson RE, Ciegler A. L-Asparaginase production by Erwinia aroideae. Journal of Applied Microbiology. 1969; 18:64-69.

29. Gordon SA, Fleck A, Bell J. Optimal conditions for the estimation of ammonium by the Berthelot reaction. Annals of Clinical Biochemistry. 1978; 1: 270-275. https://doi.org/10.1177/000456327801500164

30. Park GE, Oh HN, Ahn SY. Improvement of the ammonia analysis by the phenate method in water and wastewater. Bulletin of Korean Chemical Society. 2009; 30:2032-2038. https://doi.org/10.5012/bkcs.2009.30.9.2032

31. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. The Journal of Biological Chemistry. 1951; 193:265-275.

32. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970; 227:680-685. https://doi.org/10.1038/227680a0

33. Taddei A, Rodríguez MJ, Márquez-Vilchez E, Castelli C. Isolation and identification of Streptomyces spp. from Venezuelan soils: Morphological and biochemical studies. I. Microbiological Research. 2006; 161:222-231. https://doi.org/10.1016/j.micres.2005.08.004

34. Yadav AK, Srivastava AK, Yandigeri MS, Kashyap SK, Modi DR, Arora DK. Characterization of indigenous copper-resistant Streptomycetes from chickpea (Cicer arietinum L.) fields. Annals of Microbiology. 2010; 60:605-614. https://doi.org/10.1007/s13213-010-0095-3

35. Deshpande N, Choubey P, Agashe M. Studies on optimization of growth parameters for L-Asparaginase production by Streptomyces ginsengisoli. The Scientific World Journal. 2014; 1-6. https://doi.org/10.1155/2014/895167

36. Kimble KW, Walker JP, Finegold DN, Asher SA. Progress towards the development of a point-of-care photonic crystal ammonia sensor. Analytical and Bioanalytical Chemistry. 2006; 385:678-685. https://doi.org/10.1007/s00216-006-0453-y

37. Thaer TA, Ellaiah P. L-Asparaginase production by a Streptomycete and optimization of production parameters. Journal of Pharmaceutical and Biomedical Sciences. 2013; 29:859- 869.

38. Prista AA, Kyridio DA. L-Asparaginase of Thermus thermophilus: purification, properties and identification of essential amino acids for catalytic activity. Molecular and Cellular Biochemistry. 2001; 216:93-101. https://doi.org/10.1023/A:1011066129771

39. Price NC, Stevens L. Fundamentals of Enzymology. Third Edition. Oxford: Oxford University Press; 2000.

40. Bull AT, Bushnel ME. Environmental control of fungal growth. In: Smith, JE and Berry DE editors. The filamentous fungi: Edward Arnold, London; 1975, p. 1-26.

41. Kumar S, Dasu VV, Pakshirajan K. Purification and characterization of sparaginas-free L-Asparaginase from Pectobacterium carotovorum MTCC 1428. Bioresource Technology. 2011; 102:2077-2082. https://doi.org/10.1016/j.biortech.2010.07.114

42. Willis RC, Woolfolk R. Asparagine utilization in E. coli. Journal of Bacteriology. 1974; 118:231-241.

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