Research Article
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Cloning and Purification of L-Asparaginase from Enterobacter carcerogenus

Year 2022, Volume: 12 Issue: 1, 455 - 463, 01.03.2022
https://doi.org/10.21597/jist.962862

Abstract

In this study, the gene coding for EcL-ASP from Enterobacter carcerogenus was identified in full sequence and cloned into a mesophilic organism. The gene encoding L-asparaginase was transferred to the pET-28a (+) vector to ensure its expression in Escherichia coli BL21 (DE3) pLysS. The recombinant protein containing the N-terminal histidine tail (6xHis tag) was purified by Nickel affinity chromatography. As a result of SDS-PAGE, it was determined that the purified protein consisted of a single type of polypeptide. In the theoretical calculation, the subunit molecular weight of the recombinant protein containing the histidine tail was found to be 37 kDa. It was observed that the cloned enzyme had low L-glutaminase activity. The pH and temperature at which the recombinant enzyme showed the best activity were determined as 7.0 and 37 °C, respectively. From the drawn Lineweaver-Burk graph, it is estimated that the Km value is 0.06 mM and the Vmax value is 666.7 U mg-1 protein.

Supporting Institution

KTU-BAP

Project Number

5615

Thanks

This work was supported by KTU-BAP (Project number is FBA-2016-5615). The authors wish to thank Prof.Dr. Sabriye Çanakçı due to her contribution in providing bacteria.

References

  • Amena S, Vishalakshi N, Prabhakar M, Dayanand A, Lingappa K, 2010. Production, purification and characterization of l-asparaginase from Streptomyces gulbargensis. Brazilian Journal of Microbiology 41:173-178.
  • Anonymous, 2021. ProtParam tool. https://web.expasy.org/protparam/(Date of access: 07 May 2021)
  • Borek D, Jaskolski M, 2001. Sequence analysis of enzymes with asparaginase activity. Acta biochimica Polonica 48:893-902.
  • Dhanam JG, Kannan S, 2013. L-asparaginase: types perspectives and applications. Advance Biology Technology 13:1-5.
  • Eisele N, Linke D, Bitzer K, Na'amnieh S, Nimtz M, Berger RG, 2011. The first characterized asparaginase from a basidiomycete, Flammulina velutipes. Bioresource Technology 102:3316-3321.
  • Hammes GG, 2008. How do enzymes really work? Journal of Biological Chemistry 283(33):22337-22346.
  • Joner PE, 1976. Purification and properties of l-asparaginase b from Acinetobacter calcoaceticus. Biochimica et biophysica acta 438:287-295.
  • Kacagan M, Inan K, Belduz AO, Canakci S, 2013. Flavobacterium anatoliense sp nov., isolated from fresh water, and emended description of Flavobacterium ceti. International Journal of Systematic and Evolutionary Microbiology 63:2075-2081.
  • Kamble VP, Rao RS, Borkar PS, Khobragade CN, Dawane BS, 2006. 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 and Biophysics 43:391-394.
  • Khushoo A, Pal Y, Singh BN, Mukherjee KJ, 2004. Extracellular expression and single step purification of recombinant Escherichia coli L-asparaginase II. Protein Expression and Purification 38:29-36.
  • Kolcuoglu Y, Colak A, Faiz O, Belduz AO, 2010. Cloning, expression and characterization of highly thermo- and pH-stable maltogenic amylase from a thermophilic bacterium Geobacillus caldoxylosilyticus TK4. Process Biochemistry 45:821-828.
  • Kotzia GA, Labrou NE, 2007. L-asparaginase from Erwinia chrysanthemi 3937: Cloning, expression and characterization. Journal of Biotechnology 127:657-669.
  • Kumar K, Verma N, 2012. The various sources and application of L-asparaginase. Asian Journal of Biochemical and Pharmaceutical Research 3:197-205.
  • Lingnert H, Grivas S, Jägerstad M, Skog K, Törnqvist M, Åman P, 2002. Acrylamide in food: mechanisms of formation and influencing factors during heating of foods. Scandinavian Journal of Nutrition 46:159-172.
  • Manna S, Sinha A, Sadhukhan R, Chakrabarty SL, 1995. Purification, characterization and antitumor-activity of l-asparaginase isolated from Pseudomonas stutzeri Mb-405. Current Microbiology 30:291-298.
  • Mario S, Sandra K, Igor P,2007. Structure, Substrate Complexation and Reaction Mechanism of Bacterial Asparaginases. Current Chemical Biology 1:75-86.
  • Mesas JM, Gil JA, Martin JF, 1990. Characterization and partial-purification of l-asparaginase from Corynebacterium glutamicum. Journal of General Microbiology 136:515-519.
  • Mottram DS, Wedzicha BL, Dodson AT, 2002. Food chemistry: Acrylamide is formed in the Maillard reaction. Nature 419:448-449.
  • Patro KKR, Satpathy S, Gupta N, 2011. Evaluation of some fungi for L-asparaginase production. Indian Journal Of Fundamental And Applied Life Sciences 1:219-221.
  • Pedreschi F, Kaack K, Granby K, 2008. The effect of asparaginase on acrylamide formation in French fries. Food Chemistry 109:386-392.
  • Savitri NA, Azmi W, 2003. Microbial L-asparaginase: a potent antitumor enzyme. Indian Journal of Biotechnology 2:184-194.
  • Singh P, Banik RM, 2013. Biochemical Characterization and Antitumor Study of L-Glutaminase from Bacillus cereus MTCC 1305. Applied Biochemistry and Biotechnology 171:522-531.
  • Singh Y, Gundampati RK, Jagannadham MV, Srivastava SK, 2013. Extracellular l-Asparaginase from a Protease-Deficient Bacillus aryabhattai ITBHU02: Purification, Biochemical Characterization, and Evaluation of Antineoplastic Activity In Vitro. Applied Biochemistry and Biotechnology 171:1759-1774.
  • Sudhir AP, Dave BR, Prajapati AS, Panchal K, Patel D, 2014. Subramanian, R. B., Characterization of a Recombinant Glutaminase-Free L-Asparaginase (ansA3) Enzyme with High Catalytic Activity from Bacillus licheniformis. Applied Biochemistry and Biotechnology 174:2504-2515.
  • Vidya J, Pandey A, 2012. Recombinant Expression and Characterization of L-Asparaginase II from a Moderately Thermotolerant Bacterial Isolate. Applied Biochemistry and Biotechnology 167:973-980.
  • Zhang Y, Li D, Li Y, 2017. Expression and purification of L-asparaginase from Escherichia coli and the inhibitory effects of cyclic dipeptides. Natural Product Research 18:2099-2106.
  • Zuo S, Xue D, Zhang T, Jiang B, Mu W, 2014. Biochemical characterization of an extremely thermostablel-asparaginase from Thermococcus gammatolerans EJ3. Journal of Molecular Catalysis B: Enzymatic 109:122-129.
Year 2022, Volume: 12 Issue: 1, 455 - 463, 01.03.2022
https://doi.org/10.21597/jist.962862

Abstract

Project Number

5615

References

  • Amena S, Vishalakshi N, Prabhakar M, Dayanand A, Lingappa K, 2010. Production, purification and characterization of l-asparaginase from Streptomyces gulbargensis. Brazilian Journal of Microbiology 41:173-178.
  • Anonymous, 2021. ProtParam tool. https://web.expasy.org/protparam/(Date of access: 07 May 2021)
  • Borek D, Jaskolski M, 2001. Sequence analysis of enzymes with asparaginase activity. Acta biochimica Polonica 48:893-902.
  • Dhanam JG, Kannan S, 2013. L-asparaginase: types perspectives and applications. Advance Biology Technology 13:1-5.
  • Eisele N, Linke D, Bitzer K, Na'amnieh S, Nimtz M, Berger RG, 2011. The first characterized asparaginase from a basidiomycete, Flammulina velutipes. Bioresource Technology 102:3316-3321.
  • Hammes GG, 2008. How do enzymes really work? Journal of Biological Chemistry 283(33):22337-22346.
  • Joner PE, 1976. Purification and properties of l-asparaginase b from Acinetobacter calcoaceticus. Biochimica et biophysica acta 438:287-295.
  • Kacagan M, Inan K, Belduz AO, Canakci S, 2013. Flavobacterium anatoliense sp nov., isolated from fresh water, and emended description of Flavobacterium ceti. International Journal of Systematic and Evolutionary Microbiology 63:2075-2081.
  • Kamble VP, Rao RS, Borkar PS, Khobragade CN, Dawane BS, 2006. 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 and Biophysics 43:391-394.
  • Khushoo A, Pal Y, Singh BN, Mukherjee KJ, 2004. Extracellular expression and single step purification of recombinant Escherichia coli L-asparaginase II. Protein Expression and Purification 38:29-36.
  • Kolcuoglu Y, Colak A, Faiz O, Belduz AO, 2010. Cloning, expression and characterization of highly thermo- and pH-stable maltogenic amylase from a thermophilic bacterium Geobacillus caldoxylosilyticus TK4. Process Biochemistry 45:821-828.
  • Kotzia GA, Labrou NE, 2007. L-asparaginase from Erwinia chrysanthemi 3937: Cloning, expression and characterization. Journal of Biotechnology 127:657-669.
  • Kumar K, Verma N, 2012. The various sources and application of L-asparaginase. Asian Journal of Biochemical and Pharmaceutical Research 3:197-205.
  • Lingnert H, Grivas S, Jägerstad M, Skog K, Törnqvist M, Åman P, 2002. Acrylamide in food: mechanisms of formation and influencing factors during heating of foods. Scandinavian Journal of Nutrition 46:159-172.
  • Manna S, Sinha A, Sadhukhan R, Chakrabarty SL, 1995. Purification, characterization and antitumor-activity of l-asparaginase isolated from Pseudomonas stutzeri Mb-405. Current Microbiology 30:291-298.
  • Mario S, Sandra K, Igor P,2007. Structure, Substrate Complexation and Reaction Mechanism of Bacterial Asparaginases. Current Chemical Biology 1:75-86.
  • Mesas JM, Gil JA, Martin JF, 1990. Characterization and partial-purification of l-asparaginase from Corynebacterium glutamicum. Journal of General Microbiology 136:515-519.
  • Mottram DS, Wedzicha BL, Dodson AT, 2002. Food chemistry: Acrylamide is formed in the Maillard reaction. Nature 419:448-449.
  • Patro KKR, Satpathy S, Gupta N, 2011. Evaluation of some fungi for L-asparaginase production. Indian Journal Of Fundamental And Applied Life Sciences 1:219-221.
  • Pedreschi F, Kaack K, Granby K, 2008. The effect of asparaginase on acrylamide formation in French fries. Food Chemistry 109:386-392.
  • Savitri NA, Azmi W, 2003. Microbial L-asparaginase: a potent antitumor enzyme. Indian Journal of Biotechnology 2:184-194.
  • Singh P, Banik RM, 2013. Biochemical Characterization and Antitumor Study of L-Glutaminase from Bacillus cereus MTCC 1305. Applied Biochemistry and Biotechnology 171:522-531.
  • Singh Y, Gundampati RK, Jagannadham MV, Srivastava SK, 2013. Extracellular l-Asparaginase from a Protease-Deficient Bacillus aryabhattai ITBHU02: Purification, Biochemical Characterization, and Evaluation of Antineoplastic Activity In Vitro. Applied Biochemistry and Biotechnology 171:1759-1774.
  • Sudhir AP, Dave BR, Prajapati AS, Panchal K, Patel D, 2014. Subramanian, R. B., Characterization of a Recombinant Glutaminase-Free L-Asparaginase (ansA3) Enzyme with High Catalytic Activity from Bacillus licheniformis. Applied Biochemistry and Biotechnology 174:2504-2515.
  • Vidya J, Pandey A, 2012. Recombinant Expression and Characterization of L-Asparaginase II from a Moderately Thermotolerant Bacterial Isolate. Applied Biochemistry and Biotechnology 167:973-980.
  • Zhang Y, Li D, Li Y, 2017. Expression and purification of L-asparaginase from Escherichia coli and the inhibitory effects of cyclic dipeptides. Natural Product Research 18:2099-2106.
  • Zuo S, Xue D, Zhang T, Jiang B, Mu W, 2014. Biochemical characterization of an extremely thermostablel-asparaginase from Thermococcus gammatolerans EJ3. Journal of Molecular Catalysis B: Enzymatic 109:122-129.
There are 27 citations in total.

Details

Primary Language English
Journal Section Moleküler Biyoloji ve Genetik / Moleculer Biology and Genetic
Authors

Yakup Kolcuoğlu 0000-0002-4611-2433

Ümmühan Çakmak 0000-0001-8719-2436

Project Number 5615
Publication Date March 1, 2022
Submission Date July 5, 2021
Acceptance Date October 21, 2021
Published in Issue Year 2022 Volume: 12 Issue: 1

Cite

APA Kolcuoğlu, Y., & Çakmak, Ü. (2022). Cloning and Purification of L-Asparaginase from Enterobacter carcerogenus. Journal of the Institute of Science and Technology, 12(1), 455-463. https://doi.org/10.21597/jist.962862
AMA Kolcuoğlu Y, Çakmak Ü. Cloning and Purification of L-Asparaginase from Enterobacter carcerogenus. J. Inst. Sci. and Tech. March 2022;12(1):455-463. doi:10.21597/jist.962862
Chicago Kolcuoğlu, Yakup, and Ümmühan Çakmak. “Cloning and Purification of L-Asparaginase from Enterobacter Carcerogenus”. Journal of the Institute of Science and Technology 12, no. 1 (March 2022): 455-63. https://doi.org/10.21597/jist.962862.
EndNote Kolcuoğlu Y, Çakmak Ü (March 1, 2022) Cloning and Purification of L-Asparaginase from Enterobacter carcerogenus. Journal of the Institute of Science and Technology 12 1 455–463.
IEEE Y. Kolcuoğlu and Ü. Çakmak, “Cloning and Purification of L-Asparaginase from Enterobacter carcerogenus”, J. Inst. Sci. and Tech., vol. 12, no. 1, pp. 455–463, 2022, doi: 10.21597/jist.962862.
ISNAD Kolcuoğlu, Yakup - Çakmak, Ümmühan. “Cloning and Purification of L-Asparaginase from Enterobacter Carcerogenus”. Journal of the Institute of Science and Technology 12/1 (March 2022), 455-463. https://doi.org/10.21597/jist.962862.
JAMA Kolcuoğlu Y, Çakmak Ü. Cloning and Purification of L-Asparaginase from Enterobacter carcerogenus. J. Inst. Sci. and Tech. 2022;12:455–463.
MLA Kolcuoğlu, Yakup and Ümmühan Çakmak. “Cloning and Purification of L-Asparaginase from Enterobacter Carcerogenus”. Journal of the Institute of Science and Technology, vol. 12, no. 1, 2022, pp. 455-63, doi:10.21597/jist.962862.
Vancouver Kolcuoğlu Y, Çakmak Ü. Cloning and Purification of L-Asparaginase from Enterobacter carcerogenus. J. Inst. Sci. and Tech. 2022;12(1):455-63.