Isolation, molecular identification and production investigation of Bacillus-producing L-Asparaginase from Jiroft microflora
Subject Areas : Microbial BiotechnologyArastoo Badoei-dalfard 1 , Zahra Karami 2 , Narjes Ramezani-pour 3
1 - Associate Professors, Department of Biology, Faculty of Sciences, Shahid Bahonar University of Kerman, Kerman, Iran.
2 - Assistant Professors, Department of Biology, Faculty of Sciences, Shahid Bahonar University of Kerman, Kerman, Iran.
3 - M.Sc., Department of Biology, Faculty of Sciences, Shahid Bahonar University of Kerman, Kerman, Iran.
Keywords: Screening, Bacillus, L-asparaginase,
Abstract :
Background & Objectives: Cancer cells are not capable of producing L-asparagine, and mainly are depended to the L-asparagine from the circulating plasma pools. L-Asparaginase (L-asparagine amidohydrolase EC 3.5.1.1) catalysis the conversion of L-asparagine to L-aspartate and ammonium. L-asparaginase is the first enzyme with anti-leukemic activity to be intensively studied in human beings. Isolation and identification of L-asparaginase producing bacteria from Jiroft microflora was the aim of this study. Materials & Methods: In this sectional study, soil and water samples were collected from citrus orchards and waste of milk factory in Jiroft, Kerman province. The bacterial-producing L-asparaginase was screened on agar medium supplied with L-asparagine and phenol red indicator dye. L-asparaginase activity was detected on the basis of pink color around the colony. The best strains were finally identified using PCR method. Results: 16S rRNA sequencing results showed that the best isolate-producing L-asparaginase belonged to the Bacillus genus. DJK23 strain produced the maximum L-asparaginase, approximately 165 U/ml. The our analysis showed that L-asparaginase was active in a pH rang of 5 to 8, with maximal enzyme activity at pH 7. It is mentioned that this enzyme retains 36% of its initial activity at pH 5. Conclusion: Although L-asparaginase from bacteria has been extensively characterized, a similar attention has not been paid to Bacillus. These results showed that Bacillus L-Asparaginase has desirable activity in the presence of neutral and acidic pH, which can be considered as a therapeutic and industrial enzyme.
1. Keating M, Holmes R, Lerner S, Ho D. L-asparaginase and PEG asparaginase-past, present, and future. Leukemia & Lymphoma. 1993; 10: 153-157.
2. Muller HJ, Boos J. Use of L-asparaginase in childhood ALL. Crit Rev Oncol Hematol. 1998; 28: 13-97.
3. Roberts J, Prager M, Bachynsky N. The antitumor activity of Escherchia coli L-asparaginase. Cancer Res. 1966; 26: 2213-2217.
4. Sanjay K, Pakshirajan K, Venkata DV. Development of medium for enhanced production of glutaminase-free L-asparaginase from Pectobacterium carotovorum MTCC 1428. Appl Microbiol Biotechnol. 2009; 84: 477-486.
5. Arya LS. Childhood cancer-challenges and opportunities. Indian J Pediatr. 2003; 70: 159-162.
6. Broome JD. Evidence that the L-asparaginase of guinea pig serum is responsible for its antilymphoma effects and properties of the L-Asparaginase of pig serum in relation to those of antilymphoma substances. J Exp Med. 1963; 118: 99-120.
7. Fisher SH, Wray LV. Bacillus subtilis 168 contains two differentially regulated genes encoding Lasparaginase. J Bacteriol. 2002; 184: 2148-2154.
8. Mesas JM, Gil JA, Martin JF. Characterization and partial purification of L-asparaginase from Corynebacterium glutamicum. J Gen Microbiol. 1990; 136: 515-519.
9. Pieters R, Appel I, Kuehnel HJ, Tetzlaff-Fohr I, Pichlmeier U, van der Vaart I, Visser E, Stigter R. Pharmacokinetics, pharmacodynamics, efficacy and safety of a new recombinant asparaginase preparation in children with previously untreated acute lymphoblastic leukemia: a randomized phase 2 clinical trial. Blood. 2008; 112(13): 4832-4838.
10. Georgia AK, Nikolaos EL. L-Asparaginase from Erwinia Chrysanthemi 3937: cloning, expression and characterization. J Bacteriol. 2007; 127: 657-669.
11. Prista AA, Kyridio DA. L-asparaginase of Thermus thermophilus: purification, properties and identification of essential amino acids for catalytic activity. Mol Cell Biochem. 2001; 216: 93-101.
12. Amardeep K, Yogender P, Mukherjee KJ. Optimization of extracellular production of recombinant asparaginase in Escherichia coli in shake-flask and bioreactor. Appl Microbial Biotechnol. 2005; 68: 189-197.
13. Ashraf AEB, Mohamed S, Jehan M. Production, isolation, and purification of L-asparaginase from Pseudomonas aeruginosa 50071 using solid-state fermentation. J Biochem Mol Biol. 2004; 37: 387-393.
14. Dhevendaran K, Anithakumari YK. L-asparaginase activity in growing conditions of Streptomyces sp. associated with Therapon jarbua and Villorita cyprinoids of Veli lake, South India. Fish Technol. 2002; 39: 155-159.
15. Geckil H, Gencer S. Production of L-asparaginase in Enterobacter aerogenes expressing Vitreoscillahemoglobin for efficient oxygen uptake. Appl Microbiol Biotechnol. 2004; 63: 691-697.
16. Pinheiro IO, Araujo JM, Ximenes ECPA, Pinto JCS, Alves TLM. Production of L-asparaginase by Zymomonas mobilis strain CP4. Biomaterial Diagnostic. 2001; 6: 243-244.
17. Savitri N, Asthana AW. Microbial L-asparaginase: a potent antitumor enzyme. Indian J Biotechnol. 2003; 2: 184-194.
18. Imada A, Igarasi S, Nakahama K, Isono M. Asparaginase and glutaminase activities of microorganisms. J Gen Microbiol. 1973; 76: 85-99.
19. Ramezani–pour N, Badoei-Dalfard A, Namaki-Shoushtari A, Karami Z. Nitrile-metabolizing potential of Bacillus cereus strain FA12; Nitrilase production, purification and characterization. Biocatal Biotransform. 2015; 33: 156-166.
20. Badoei-Dalfard A, Karami Z. Screening and isolation of an organic solvent tolerant-protease from Bacillus sp. JER02: Activity optimization by response surface methodology. J Mol Catal B: Enzym. 2013; 89: 15-23.
21. Tamura K, Dudley J, Nei M, Kumar S. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Bio Evol. 2007; 24: 1596-1599.
22. Badoei-Dalfard A. Purification and characterization of L-asparaginase from Pseudomonas aeruginosa strain SN004: Production optimization by statistical methods. Biocatal Agric Biotechnol. 2015; 4: 388-397.
23. Gholamian S, Gholamian S, Nazemi A, Nargesi MM. Optimization of culture media for L-asparaginase production by newly isolated bacteria, Bacillus sp. GH5. Microbiol. 2013; 82 (6): 856-863.
24. Hymavathi M, Sathish T, Subba RC, Prakasham RS. Enhancement of L-asparaginase production by isolated Bacillus circulans (MTCC 8574) using response surface methodology. Appl Microbiol Biotechnol. 2009; 159: 191-198.
25. Mahajan RV, Kumar V, Rajendran V, Saran S, Ghosh PC, Saxena RK. Purification and characterization of a novel and robust L-asparaginase having low-glutaminase activity from Bacillus licheniformis: in vitro evaluation of anti-cancerous properties. PLoS ONE. 2014; 9(6): e99037.
26. Mahajana RV, Saranb S, Kameswarana K, Kumara V, Saxena RK. Efficient production of L-asparaginase from Bacillus licheniformis with low-glutaminase activity: Optimization, scale up and acrylamide degradation studies. Bioresour Technol. 2012; 125: 11-16.
27. Wafaa KH, Maysa EM. L-asparaginase hyperproducing recombinant Bacillus strains obtained by interspecific protoplast fusion. J Genet Eng Biotech. 2010; 8(2): 67-74.
28. Gulati R, Saxena RK, Gupta R. A rapid plate assay for screening L-asparaginase producing micro-organisms. Lett Appl Microbiol. 1997; 24(1): 23-26.
29. Audipudi AV, Pallavi R, Supriya G. Characterization of L-asparaginase producing bacteria from Mangrove Soil. Int J Chem Tech Res. 2013; 5(1): 109-112.
30. Pradhan B, Dash SK, Sahoo S. Screening and characterization of extracelluar L-asparaginase producing Bacillus subtilis strain hswx88, isolated from Taptapani hot spring of Odisha, India. Asian Pac J Trop Biomed. 2013; 3(12): 936-941.
31. 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. Trop J Pharm Res. 2009; 8(4): 353-360.
32. Kumar S, Dasu VV, Pakshirajan K. Localization and production of novel L-asparaginase from Pectobacterium carotovorum MTCC 1428. Process Biochem. 2010; 45: 223-229.
33. Elshafei MA, Mohamed HM, Abd-Elmontasr AM, Mahmoud DA, Elghonemy DH. Purification, characterization and antitumor activity of L-asparaginase from Penicillium brevicompactum NRC 829. Br Microb Res J. 2012; 2(3): 158-174.
34. Khamna S, Yokota A, Lumyong S. L-asparaginase production by actinomycetes isolated from some Thai medicinal plant rhizosphere soils. Int J Integr Biol. 2009; 6(1): 22-26.
_||_1. Keating M, Holmes R, Lerner S, Ho D. L-asparaginase and PEG asparaginase-past, present, and future. Leukemia & Lymphoma. 1993; 10: 153-157.
2. Muller HJ, Boos J. Use of L-asparaginase in childhood ALL. Crit Rev Oncol Hematol. 1998; 28: 13-97.
3. Roberts J, Prager M, Bachynsky N. The antitumor activity of Escherchia coli L-asparaginase. Cancer Res. 1966; 26: 2213-2217.
4. Sanjay K, Pakshirajan K, Venkata DV. Development of medium for enhanced production of glutaminase-free L-asparaginase from Pectobacterium carotovorum MTCC 1428. Appl Microbiol Biotechnol. 2009; 84: 477-486.
5. Arya LS. Childhood cancer-challenges and opportunities. Indian J Pediatr. 2003; 70: 159-162.
6. Broome JD. Evidence that the L-asparaginase of guinea pig serum is responsible for its antilymphoma effects and properties of the L-Asparaginase of pig serum in relation to those of antilymphoma substances. J Exp Med. 1963; 118: 99-120.
7. Fisher SH, Wray LV. Bacillus subtilis 168 contains two differentially regulated genes encoding Lasparaginase. J Bacteriol. 2002; 184: 2148-2154.
8. Mesas JM, Gil JA, Martin JF. Characterization and partial purification of L-asparaginase from Corynebacterium glutamicum. J Gen Microbiol. 1990; 136: 515-519.
9. Pieters R, Appel I, Kuehnel HJ, Tetzlaff-Fohr I, Pichlmeier U, van der Vaart I, Visser E, Stigter R. Pharmacokinetics, pharmacodynamics, efficacy and safety of a new recombinant asparaginase preparation in children with previously untreated acute lymphoblastic leukemia: a randomized phase 2 clinical trial. Blood. 2008; 112(13): 4832-4838.
10. Georgia AK, Nikolaos EL. L-Asparaginase from Erwinia Chrysanthemi 3937: cloning, expression and characterization. J Bacteriol. 2007; 127: 657-669.
11. Prista AA, Kyridio DA. L-asparaginase of Thermus thermophilus: purification, properties and identification of essential amino acids for catalytic activity. Mol Cell Biochem. 2001; 216: 93-101.
12. Amardeep K, Yogender P, Mukherjee KJ. Optimization of extracellular production of recombinant asparaginase in Escherichia coli in shake-flask and bioreactor. Appl Microbial Biotechnol. 2005; 68: 189-197.
13. Ashraf AEB, Mohamed S, Jehan M. Production, isolation, and purification of L-asparaginase from Pseudomonas aeruginosa 50071 using solid-state fermentation. J Biochem Mol Biol. 2004; 37: 387-393.
14. Dhevendaran K, Anithakumari YK. L-asparaginase activity in growing conditions of Streptomyces sp. associated with Therapon jarbua and Villorita cyprinoids of Veli lake, South India. Fish Technol. 2002; 39: 155-159.
15. Geckil H, Gencer S. Production of L-asparaginase in Enterobacter aerogenes expressing Vitreoscillahemoglobin for efficient oxygen uptake. Appl Microbiol Biotechnol. 2004; 63: 691-697.
16. Pinheiro IO, Araujo JM, Ximenes ECPA, Pinto JCS, Alves TLM. Production of L-asparaginase by Zymomonas mobilis strain CP4. Biomaterial Diagnostic. 2001; 6: 243-244.
17. Savitri N, Asthana AW. Microbial L-asparaginase: a potent antitumor enzyme. Indian J Biotechnol. 2003; 2: 184-194.
18. Imada A, Igarasi S, Nakahama K, Isono M. Asparaginase and glutaminase activities of microorganisms. J Gen Microbiol. 1973; 76: 85-99.
19. Ramezani–pour N, Badoei-Dalfard A, Namaki-Shoushtari A, Karami Z. Nitrile-metabolizing potential of Bacillus cereus strain FA12; Nitrilase production, purification and characterization. Biocatal Biotransform. 2015; 33: 156-166.
20. Badoei-Dalfard A, Karami Z. Screening and isolation of an organic solvent tolerant-protease from Bacillus sp. JER02: Activity optimization by response surface methodology. J Mol Catal B: Enzym. 2013; 89: 15-23.
21. Tamura K, Dudley J, Nei M, Kumar S. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Bio Evol. 2007; 24: 1596-1599.
22. Badoei-Dalfard A. Purification and characterization of L-asparaginase from Pseudomonas aeruginosa strain SN004: Production optimization by statistical methods. Biocatal Agric Biotechnol. 2015; 4: 388-397.
23. Gholamian S, Gholamian S, Nazemi A, Nargesi MM. Optimization of culture media for L-asparaginase production by newly isolated bacteria, Bacillus sp. GH5. Microbiol. 2013; 82 (6): 856-863.
24. Hymavathi M, Sathish T, Subba RC, Prakasham RS. Enhancement of L-asparaginase production by isolated Bacillus circulans (MTCC 8574) using response surface methodology. Appl Microbiol Biotechnol. 2009; 159: 191-198.
25. Mahajan RV, Kumar V, Rajendran V, Saran S, Ghosh PC, Saxena RK. Purification and characterization of a novel and robust L-asparaginase having low-glutaminase activity from Bacillus licheniformis: in vitro evaluation of anti-cancerous properties. PLoS ONE. 2014; 9(6): e99037.
26. Mahajana RV, Saranb S, Kameswarana K, Kumara V, Saxena RK. Efficient production of L-asparaginase from Bacillus licheniformis with low-glutaminase activity: Optimization, scale up and acrylamide degradation studies. Bioresour Technol. 2012; 125: 11-16.
27. Wafaa KH, Maysa EM. L-asparaginase hyperproducing recombinant Bacillus strains obtained by interspecific protoplast fusion. J Genet Eng Biotech. 2010; 8(2): 67-74.
28. Gulati R, Saxena RK, Gupta R. A rapid plate assay for screening L-asparaginase producing micro-organisms. Lett Appl Microbiol. 1997; 24(1): 23-26.
29. Audipudi AV, Pallavi R, Supriya G. Characterization of L-asparaginase producing bacteria from Mangrove Soil. Int J Chem Tech Res. 2013; 5(1): 109-112.
30. Pradhan B, Dash SK, Sahoo S. Screening and characterization of extracelluar L-asparaginase producing Bacillus subtilis strain hswx88, isolated from Taptapani hot spring of Odisha, India. Asian Pac J Trop Biomed. 2013; 3(12): 936-941.
31. 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. Trop J Pharm Res. 2009; 8(4): 353-360.
32. Kumar S, Dasu VV, Pakshirajan K. Localization and production of novel L-asparaginase from Pectobacterium carotovorum MTCC 1428. Process Biochem. 2010; 45: 223-229.
33. Elshafei MA, Mohamed HM, Abd-Elmontasr AM, Mahmoud DA, Elghonemy DH. Purification, characterization and antitumor activity of L-asparaginase from Penicillium brevicompactum NRC 829. Br Microb Res J. 2012; 2(3): 158-174.
34. Khamna S, Yokota A, Lumyong S. L-asparaginase production by actinomycetes isolated from some Thai medicinal plant rhizosphere soils. Int J Integr Biol. 2009; 6(1): 22-26.
