جداسازی، شناسایی مولکولی و بررسی میزان تولید سویه های باسیلوس مولد آنزیم آسپاراژیناز از زیست بوم شهرستان جیرفت
محورهای موضوعی : زیست فناوری میکروبی
ارسطو بدوئی دلفارد
1
(دانشیار، دانشگاه شهید باهنر کرمان، گروه زیست شناسی)
زهرا کرمی
2
(استادیار، دانشگاه شهید باهنر کرمان، گروه زیست شناسی)
نرجس رمضانی پور
3
(کارشناس ارشد، دانشگاه شهید باهنر کرمان، گروه زیست شناسی)
کلید واژه: باسیلوس, L-آسپاراژیناز, غربالگری,
چکیده مقاله :
سابقه و هدف: سلول های سرطانی قادر به تولید L-آسپاراژین نمی باشند و به L-آسپاراژین پلاسمای خون وابسته هستند. L-آسپاراژیناز (L-آسپاراژین آمیدوهیدرولاز EC 3.5.1.1) باعث تبدیل L-آسپاراژین به آسپاراتات و آمونیاک می شود. L-آسپاراژیناز اولین آنزیم با فعالیت ضد سرطان خون می باشد که به طور گسترده در انسان مورد بررسی قرار گرفته است. این مطالعه با هدف جداسازی و شناسایی سویه های مولد آنزیم آسپاراژیناز از زیست بوم شهرستان جیرفت انجام شد. مواد و روش ها: در این مطالعه مقطعی، نمونه های آب و خاک از باغات مرکبات و فاضلاب کارخانه شیر شهرستان جیرفت در استان کرمان جمع آوری گردید. باکتری های تولید کننده L-آسپاراژیناز بر روی محیط جامد حاوی سوبسترای آسپاراژین و معرف فنل رد غربالگری شدند. میزان فعالیت آسپاراژینازی بر اساس قطر هاله ایجاد شده اندازه گیری گردید. بهترین سویه های انتخاب شده با روش مولکولی PCR شناسایی شدند. یافته ها: نتایج تعیین توالی ژن16S rRNA نشان داد که سویه های برتر متعلق به جنس باسیلوس می باشند. بیشترین میزان تولید آسپاراژیناز توسط سویه DJK23 حدود 165 واحد بر میلی لیتر بود. نتایج نشان داد که L-آسپاراژیناز در اسیدیته 5 تا 8 فعالیت دارد و بیشترین فعالیت آنزیمی در اسیدیته 7 می باشد. همچنین این آنزیم در اسیدیته 5 حدود 36 درصد فعالیت آنزیمی از خود نشان داد. نتیجه گیری: با وجود قابلیت باسیلوس ها در تولید تجاری آنزیم ها، L-آسپاراژیناز این باکتری ها کمتر مورد بررسی قرار گرفته است. یافته های ما نشان داد که L-آسپاراژیناز باسیلوس فعالیت قابل توجهی در حضور اسیدیته خنثی و اسیدی نشان می دهد. بنابراین می تواند به عنوان یک آنزیم دارویی و صنعتی مورد بررسی بیشتری قرار گیرد.
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.