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  • بهینه سازی تولید ال-آسپارژیناز از سویه بومی باسیلوس جدا شده از خاک و ارزیابی فعالیت ضد سرطانی آن

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کد مقاله : JMW-2209-2035 (R1) بازدید : 161 صفحه: 259 - 270

10.30495/jmw.2022.1967544.2035

20.1001.1.20083068.1401.15.53.2.0

نوع مقاله: پژوهشی

بهینه سازی تولید ال-آسپارژیناز از سویه بومی باسیلوس جدا شده از خاک و ارزیابی فعالیت ضد سرطانی آن

محورهای موضوعی : زیست فناوری میکروبی

فروغ رهنمای رودپشتی 1 (گروه زیست شناسی،دانشکده علوم پایه، دانشگاه آزاد اسلامی واحد رشت، رشت، ایران)
لیلا اسدپور 2 (گروه زیست شناسی،دانشکده علوم پایه، دانشگاه آزاد اسلامی واحد رشت، رشت، ایران)
مهدی شهریاری نور 3 (گروه زیست شناسی،دانشکده علوم پایه، دانشگاه آزاد اسلامی واحد رشت، رشت، ایران)
بهنام راستی 4 (،گروه میکروبیولوژی،دانشکده علوم پایه، دانشگاه آزاد اسلامی واحد لاهیجان، لاهیجان، ایران)
سجاد قرقانی 5 (گروه بیوانفورماتیک، مرکز تحقیقات بیوشیمی بیوفیزیک،دانشگاه تهران، تهران، ایران)

تاریخ دریافت : 1401/05/29 تاریخ پذیرش : 1401/10/10 تاریخ انتشار : 1401/12/15

کلید واژه: خاک, بهینه سازی, باسیلوس, الـ آسپاراژیناز,

چکیده مقاله :

سابقه و هدف: باکتری ها منابع مهم آنزیم عامل ضد سرطان ال-آسپارژیناز (ASNase) هستند که برای درمان لوسمی لنفوبلاستیک حاد در سراسر جهان استفاده می شود. این مطالعه با هدف تعیین ویژگی های ASNase از باکتری های جدا شده از خاک شمال ایران انجام شد. مواد و روش ها: در مطالعه حاضر، تولید ASNase توسط سویه‌ های باکتریایی جدا شده از نمونه‌ های خاک جنگلی استان گیلان، شمال ایران مورد بررسی قرار گرفت. شرایط بهینه تولید آنزیم، سینتیک، اثر فعال‌کننده‌ ها و مهارکننده‌ ها و فعالیت ضد سرطانی ال-آسپارژیناز نیمه خالص‌شده در برابر رده‌ سلولی MCF-7مورد مطالعه قرار گرفت. یافته ها: یک جدایه با قابلیت مطلوب تولید ASNase به روش تعیین توالی ژن 16S rRNAبه عنوان باسیلوس شناسایی شد. فعالیت گلوتامینازی آنزیم 5/9 برابر کمتر از فعالیت آسپاراژینازی آن بود و آنزیم با Km و Vmax به ترتیب 0/550 مولار و 35/71 میکرو مولار بر میلی‌ لیتر در دقیقه، دارای میل ترکیبی با ال-آسپاراژین بود. آنزیم ASNase مورد مطالعه در محدوده pH بین 6/5 تا 8/5 و تا دمای ۵۵ درجه سلسیوس پایدار بود. فعالیت ASNase تحت تاثیر حضور یون های فلزی Na+،K+ قرار نگرفت و یون Mg2+ به طور قابل توجهی سبب افزایش فعالیت آنزیم شد در حالی که Ca2+ فعالیت آنزیم را کاهش داد. فعالیت ضد سرطانی ال-آسپاراژیناز در برابر رده های سلولی MCF-7 با IC50 معادل ۲۱میکروگرم بر میلی لیتر شناسایی شد. نتیجه گیری: گونه باسیلوس جدا شده از خاک گیلان به عنوان گزینه‌ ای برای تولید ال-آسپارژیناز شناسایی شد و می تواند برای استفاده در صنایع دارویی و غذایی مورد مطالعه قرار گیرد.

چکیده انگلیسی:

Background and Objectives: Bacteria are one of the most important sources of L-asparaginase (ASNase) production which is used as an anticancer agent in the treatment of acute lymphoblastic leukemia worldwide. This study aimed to optimize the ASNase production by bacteria isolated from the soil in northern Iran, and to determine its anti-cancer activity.  Materials and Methods: ASNase production by bacterial strains isolated from forest soil samples in Guilan Province, northern Iran was investigated. The optimized condition of enzyme production, kinetics, effect of activators and inhibitors and anticancer activity of the partially purified L-asparaginase against MCF-7 cell lines were studied. Results: A promising ASNase producing isolate, was identified by 16S rRNA gene sequencing as Bacillus sp. The glutaminase activity of the enzyme was found to be 5.9 times lower than its asparaginase activity and the enzyme showed affinity for L-asparagine with a Km value and Vmax of 0.055M and 35.71 µM/mL/min, respectively. The current ASNase enzyme was stable from pH 6.5 to 8.5 and stable up to 55°C. ASNase activity was not significantly affected by the presence of two metal ions Na+, K+; Mg2+ showed enhancement in enzyme activity, while Ca2+ decreased it. Anticancer activity of the purified L-asparaginase was detected against MCF-7 cell lines with IC50 of 21µg/ml. Conclusion: The soil isolate Bacillus sp. was identified as a candidate for L-asparaginase production. The future prospect of this enzyme recommends its utility in pharmaceutical and food industry.  

منابع و مأخذ:

References

  1. Darvishi F, Jahanafrooz Z, Mokhtarzadeh A. Microbial L-asparaginase as a promising enzyme for treatment of various cancers. Applied Microbiology and Biotechnology. 2022 :106 (17):5335-5347.
    2.

 

2.Sindhwad P, Desai K. media optimization, Isolation and purification of L-asparaginase from marine isolate. Asian Pac. J. Health Sci. 2015; 293:72-82.

  1. Jha SK, Pasrija D, Sinha RK, Singh HR, Nigam VK, Vidyarthi AS. Microbial L-asparaginase: a review on current scenario and future prospects. International journal of pharmaceutical sciences and research. 2012;3(9):3076.
    2.
  2. Mangamuri U, Vijayalakshmi M, Ganduri VS, Rajulapati SB, Poda S. Extracellular L-asparaginase from Streptomyces labedae VSM-6: Isolation, production and optimization of culture conditions using RSM. Pharmacognosy Journal. 2017; 9(6): 932-941.
    3.
  3. Zielezinski A, Loch JI, Karlowski WM, Jaskolski M. Massive annotation of bacterial l-asparaginases reveals their puzzling distribution and frequent gene transfer events. Scientific reports. 2022 ; 12(1):15797.
    4.
  4. Safary A, Moniri R, Hamzeh-Mivehroud M, Dastmalchi S. Highly efficient novel recombinant L-asparaginase with no glutaminase activity from a new halo-thermotolerant Bacillus strain. BioImpacts: BI. 2019;9(1):15-23.
    5.
  5. 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 characterisation. Tropical Journal of Pharmaceutical Research. 2009;8 (4): 353-360.
    6.
  6. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical biochemistry. 1976 ;72(1-2):248-54.
    7.

9.Gomori G. Preparation of buffers for use in enzyme studies. In S. P. Colowick & N. O. Kaplan (Eds.), Methods in Enzymology; 1995: 138–146. New York: Academic Press.

10.El-Naggar NE, Deraz SF, El-Ewasy SM, Suddek GM. Purification, characterization and immunogenicity assessment of glutaminase free L-asparaginase from Streptomyces brollosae NEAE-115. BMC Pharmacology and Toxicology. 2018;19(1):51.

11.Kumari PV, Sankar GG, Prabhakar T, Lakshmi SS. Purification and characterization of L-asparaginase from Streptomyces griseoluteus WS3/1. Int J Pharm Sci Rev Res. 2013;23(2):198-202.

12.Monica T, Lincoln L, Niyonzima FN, Sunil SM. Isolation, purification and characterization of fungal extracellular L-asparaginase from Mucor Hiemalis. J Biocatal Biotransform 2013; 2:1-9.

13.Warangkar SC, Khobragade CN. Purification, characterization, and effect of thiol compounds on activity of the Erwinia carotovora L-asparaginase. Enzyme Res 2010; 1:1-10.

14.Alrumman SA, Mostafa YS, Al-Izran KA, Alfaifi MY, Taha TH, Elbehairi SE. Production and anticancer activity of an L-asparaginase from Bacillus licheniformis isolated from the Red Sea, Saudi Arabia. Scientific reports. 2019;9(1):1-4.

15.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 6;9(6):e99037.

16.Verma N, Kumar K, Kaur G, Anand S. L-asparaginase: a promising chemotherapeutic agent. Critical reviews in biotechnology. 2007;27(1):45-62.

17.Kumar NM, Ramasamy R, Manonmani HK. Production and optimization of l-asparaginase from Cladosporium sp. using agricultural residues in solid state fermentation. Industrial Crops and Products. 2013; 43:150-8.

18.Thenmozhi C, Sankar R, Karuppiah V, Sampathkumar P. L-asparaginase production by mangrove derived Bacillus cereus MAB5: optimization by response surface methodology. Asian Pacific journal of tropical medicine. 2011;4(6):486-91.

  1. Arjun JK, Aneesh B, Kavitha T, Hari Krishnan K. Therapeutic L-asparaginase activity of bacteria isolated from marine sediments. International Journal of Pharmaceutical Sciences and Drug Research. 2016;8(4):229-34.
    2.

20.Mostafa Y, Alrumman S, Alamri S, Hashem M, Al-izran K, Alfaifi M, Elbehairi SE, Taha T. Enhanced production of glutaminase-free L-asparaginase by marine Bacillus velezensis and cytotoxic activity against breast cancer cell lines. Electronic Journal of Biotechnology. 2019 Nov 1;42:6-15.

  1. Pradhan B, Dash SK, Sahoo S. Screening and characterization of extracelluar L-asparaginase producing Bacillus subtilis strain hswx88, isolated from Taptapani hotspring of Odisha, India. Asian Pacific journal of tropical biomedicine. 2013 Dec 1;3(12):936-41.
    2.

22.Rahimzadeh M, Poodat M, Javadpour S, Qeshmi FI, Shamsipour F. Purification, characterization and comparison between two new L-asparaginases from Bacillus PG03 and Bacillus PG04. The open biochemistry journal. 2016; 10:35-45.

  1. Gamal-Eldeen EM, El-sayed ST. Production, immobilization and anti-tumor activity of L-asparaginase of Bacillus sp. R36. J Ameri Sci. 2010;6(8):157-65.
    2.
  2. Kumar S, Dasu VV, Pakshirajan K. Purification and characterization of glutaminase-free L-asparaginase from Pectobacterium carotovorum MTCC 1428. Bioresource technology. 2011 Jan 1;102(2):2077-82.
    3.
  3. Farag AM, Hassan SW, Beltagy EA, El-Shenawy MA. Optimization of production of anti-tumor l-asparaginase by free and immobilized marine Aspergillus terreus. The Egyptian Journal of Aquatic Research. 2015;41(4):295-302.
    4.
  4. Sudhir AP, Agarwaal VV, Dave BR, Patel DH, Subramanian RB. Enhanced catalysis of L-asparaginase from Bacillus licheniformis by a rational redesign. Enzyme and Microbial Technology. 2016; 86:1-6.
    5.

27.Safary A, Moniri R, Hamzeh-Mivehroud M, Dastmalchi S. Highly efficient novel recombinant L-asparaginase with no glutaminase activity from a new halo-thermotolerant Bacillus strain. BioImpacts: BI. 2019;9(1):15-23.

28.Hassan SW, Farag AM, Beltagy EA. Purification, characterization and anticancer activity of L-asparaginase produced by marine Aspergillus terreus. J. Pure Appl. Microbiol. 2018;12(4):1845-54.

29.Moharib SA. Anticancer activity of L-Asparaginase produced from Vigna uUnguiculata. World Sci. Res. 2018; 5: 1-12.

30.Shafei MS, El-Refai HA, Mostafa H, El-Refai AM, El-Beih FM, Easa SM, Gomaa SK. Purification, characterization and kinetic properties of Penicillium cyclopium L-asparaginase: Impact of lasparaginase on acrylamide content in potato products and its cytotoxic activity. Cur. Tre. Biotech. Pharm. 2015 Apr 1;9:132-40.

_||_

References

  1. Darvishi F, Jahanafrooz Z, Mokhtarzadeh A. Microbial L-asparaginase as a promising enzyme for treatment of various cancers. Applied Microbiology and Biotechnology. 2022 :106 (17):5335-5347.
    2.

 

2.Sindhwad P, Desai K. media optimization, Isolation and purification of L-asparaginase from marine isolate. Asian Pac. J. Health Sci. 2015; 293:72-82.

  1. Jha SK, Pasrija D, Sinha RK, Singh HR, Nigam VK, Vidyarthi AS. Microbial L-asparaginase: a review on current scenario and future prospects. International journal of pharmaceutical sciences and research. 2012;3(9):3076.
    2.
  2. Mangamuri U, Vijayalakshmi M, Ganduri VS, Rajulapati SB, Poda S. Extracellular L-asparaginase from Streptomyces labedae VSM-6: Isolation, production and optimization of culture conditions using RSM. Pharmacognosy Journal. 2017; 9(6): 932-941.
    3.
  3. Zielezinski A, Loch JI, Karlowski WM, Jaskolski M. Massive annotation of bacterial l-asparaginases reveals their puzzling distribution and frequent gene transfer events. Scientific reports. 2022 ; 12(1):15797.
    4.
  4. Safary A, Moniri R, Hamzeh-Mivehroud M, Dastmalchi S. Highly efficient novel recombinant L-asparaginase with no glutaminase activity from a new halo-thermotolerant Bacillus strain. BioImpacts: BI. 2019;9(1):15-23.
    5.
  5. 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 characterisation. Tropical Journal of Pharmaceutical Research. 2009;8 (4): 353-360.
    6.
  6. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical biochemistry. 1976 ;72(1-2):248-54.
    7.

9.Gomori G. Preparation of buffers for use in enzyme studies. In S. P. Colowick & N. O. Kaplan (Eds.), Methods in Enzymology; 1995: 138–146. New York: Academic Press.

10.El-Naggar NE, Deraz SF, El-Ewasy SM, Suddek GM. Purification, characterization and immunogenicity assessment of glutaminase free L-asparaginase from Streptomyces brollosae NEAE-115. BMC Pharmacology and Toxicology. 2018;19(1):51.

11.Kumari PV, Sankar GG, Prabhakar T, Lakshmi SS. Purification and characterization of L-asparaginase from Streptomyces griseoluteus WS3/1. Int J Pharm Sci Rev Res. 2013;23(2):198-202.

12.Monica T, Lincoln L, Niyonzima FN, Sunil SM. Isolation, purification and characterization of fungal extracellular L-asparaginase from Mucor Hiemalis. J Biocatal Biotransform 2013; 2:1-9.

13.Warangkar SC, Khobragade CN. Purification, characterization, and effect of thiol compounds on activity of the Erwinia carotovora L-asparaginase. Enzyme Res 2010; 1:1-10.

14.Alrumman SA, Mostafa YS, Al-Izran KA, Alfaifi MY, Taha TH, Elbehairi SE. Production and anticancer activity of an L-asparaginase from Bacillus licheniformis isolated from the Red Sea, Saudi Arabia. Scientific reports. 2019;9(1):1-4.

15.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 6;9(6):e99037.

16.Verma N, Kumar K, Kaur G, Anand S. L-asparaginase: a promising chemotherapeutic agent. Critical reviews in biotechnology. 2007;27(1):45-62.

17.Kumar NM, Ramasamy R, Manonmani HK. Production and optimization of l-asparaginase from Cladosporium sp. using agricultural residues in solid state fermentation. Industrial Crops and Products. 2013; 43:150-8.

18.Thenmozhi C, Sankar R, Karuppiah V, Sampathkumar P. L-asparaginase production by mangrove derived Bacillus cereus MAB5: optimization by response surface methodology. Asian Pacific journal of tropical medicine. 2011;4(6):486-91.

  1. Arjun JK, Aneesh B, Kavitha T, Hari Krishnan K. Therapeutic L-asparaginase activity of bacteria isolated from marine sediments. International Journal of Pharmaceutical Sciences and Drug Research. 2016;8(4):229-34.
    2.

20.Mostafa Y, Alrumman S, Alamri S, Hashem M, Al-izran K, Alfaifi M, Elbehairi SE, Taha T. Enhanced production of glutaminase-free L-asparaginase by marine Bacillus velezensis and cytotoxic activity against breast cancer cell lines. Electronic Journal of Biotechnology. 2019 Nov 1;42:6-15.

  1. Pradhan B, Dash SK, Sahoo S. Screening and characterization of extracelluar L-asparaginase producing Bacillus subtilis strain hswx88, isolated from Taptapani hotspring of Odisha, India. Asian Pacific journal of tropical biomedicine. 2013 Dec 1;3(12):936-41.
    2.

22.Rahimzadeh M, Poodat M, Javadpour S, Qeshmi FI, Shamsipour F. Purification, characterization and comparison between two new L-asparaginases from Bacillus PG03 and Bacillus PG04. The open biochemistry journal. 2016; 10:35-45.

  1. Gamal-Eldeen EM, El-sayed ST. Production, immobilization and anti-tumor activity of L-asparaginase of Bacillus sp. R36. J Ameri Sci. 2010;6(8):157-65.
    2.
  2. Kumar S, Dasu VV, Pakshirajan K. Purification and characterization of glutaminase-free L-asparaginase from Pectobacterium carotovorum MTCC 1428. Bioresource technology. 2011 Jan 1;102(2):2077-82.
    3.
  3. Farag AM, Hassan SW, Beltagy EA, El-Shenawy MA. Optimization of production of anti-tumor l-asparaginase by free and immobilized marine Aspergillus terreus. The Egyptian Journal of Aquatic Research. 2015;41(4):295-302.
    4.
  4. Sudhir AP, Agarwaal VV, Dave BR, Patel DH, Subramanian RB. Enhanced catalysis of L-asparaginase from Bacillus licheniformis by a rational redesign. Enzyme and Microbial Technology. 2016; 86:1-6.
    5.

27.Safary A, Moniri R, Hamzeh-Mivehroud M, Dastmalchi S. Highly efficient novel recombinant L-asparaginase with no glutaminase activity from a new halo-thermotolerant Bacillus strain. BioImpacts: BI. 2019;9(1):15-23.

28.Hassan SW, Farag AM, Beltagy EA. Purification, characterization and anticancer activity of L-asparaginase produced by marine Aspergillus terreus. J. Pure Appl. Microbiol. 2018;12(4):1845-54.

29.Moharib SA. Anticancer activity of L-Asparaginase produced from Vigna uUnguiculata. World Sci. Res. 2018; 5: 1-12.

30.Shafei MS, El-Refai HA, Mostafa H, El-Refai AM, El-Beih FM, Easa SM, Gomaa SK. Purification, characterization and kinetic properties of Penicillium cyclopium L-asparaginase: Impact of lasparaginase on acrylamide content in potato products and its cytotoxic activity. Cur. Tre. Biotech. Pharm. 2015 Apr 1;9:132-40.

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