Bioinformatic characterization and production of L-asparaginase from Bacillus sp. M62 isolated from the Maras saltern, Cusco, Peru
DOI:
https://doi.org/10.15381/rpb.v30i1.22411Keywords:
L-asparaginase, Bacillus sp. halotolerant, gen ansA3, Maras salternAbstract
The aim of this study was to perform bioinformatics characterization and optimize the production of extracellular L-asparaginase from Bacillus sp. M62, isolated from the Maras salt ponds (Cusco). To achieve this, the production of L-asparaginase was verified by the change in color of modified M9 medium, containing 0.0075% bromophenol blue, at pH 7.4 and 37°C for 72 hours. Genomic DNA was extracted to amplify the 16S ribosomal genes and the ansA3 gene. The amino acid sequence encoded by the ansA3 gene was predicted using bioinformatic analysis. The production of intracellular and extracellular L-asparaginase was evaluated at different levels of glucose, L-asparagine, NaCl, and pH in modified M9 medium. Additionally, the enzymatic activities of L-asparaginase and L-glutaminase were determined by quantifying the released ammonium using the Nessler method. Bacillus sp. M62 showed the change in color of the modified M9 medium, high similarity, and evolutionary closeness to Bacillus licheniformis. The amplified ansA3 gene was found to encode for 319 amino acids, with a predicted active site pattern (GFVITHGTDTM) and 15 immunogenic sites. The production of extracellular L-asparaginase was found to be higher than intracellular L-asparaginase and was optimized from 0.37 U/mL (0.24 U/mg) to 2.15 ± 0.39 U/mL (0.63 U/mg). Finally, it was found that Bacillus sp. M62 presents extracellular L-asparaginase with minimal L-glutaminase activity.
Downloads
References
Abdelrazek NA, Elkhatib WF, Raafat MM, Aboulwafa MM. 2019. Experimental and bioinformatics study for production of l-asparaginase from Bacillus licheniformis: a promising enzyme for medical application. AMB Express. 9(1):39. https://doi.org/10.1186/s13568-019-0751-3
Abdelrazek NA, Elkhatib WF, Raafat MM, Aboulwafa MM. 2020. Production, characterization and bioinformatics analysis of L-asparaginase from a new Stenotrophomonas maltophilia EMCC2297 soil isolate. AMB Express. 10(1). https://doi.org/10.1186/s13568-020-01005-7
Aishwariyaa Lakshmi V, Sheeja L, Devi Mari S. 2020. Screening and Identification of Asparginase and Glutaminase Producing Halophilic Bacteria from Natural Saline Habitats. Int J Recent Adv Biotechnol Nanotechnol. 3(1):34–53.
Alrumman SA, Mostafa YS, Al-izran KA, Alfaifi MY, Taha TH, Elbehairi SE. 2019. Production and Anticancer Activity of an L-Asparaginase from Bacillus licheniformis Isolated from the Red Sea, Saudi Arabia. Sci Rep. 9(1):1–14. https://doi.org/10.1038/s41598-019-40512-x
Amany BAE, Wesam AH, Zakaria AM, Rabab AED. 2021. Production of Chemotherapeutic agent L-asparaginase from Gamma-Irradiated Pseudomonas aeruginosa WCHPA075019. Jordan J Biol Sci. 14(3):403–412. https://doi.org/10.54319/jjbs/140304
Arévalo-Tristancho E, Díaz LE, Cortázar JE, Valero MF. 2019. Production and characterization of L-Asparaginases of Streptomyces isolated from the Arauca riverbank (Colombia). Open Microbiol J. 13(1):204–215. https://doi.org/10.2174/1874285801913010204
Badoei-Dalfard A. 2015. Purification and characterization of l-asparaginase from Pseudomonas aeruginosa strain SN004: Production optimization by statistical methods. Biocatal Agric Biotechnol. 4(3):388–397. https://doi.org/10.1016/j.bcab.2015.06.007
Bhargavi M, Jayamadhuri R. 2016. Isolation and screening of marine bacteria producing anti-cancer enzyme L-asparaginase. Am J Mar Sci. 4(1):1–3. https://doi.org/10.12691/marine-4-1-1
Cachumba JJM, Antunes FAF, Peres GFD, Brumano LP, Santos JC Dos, Da Silva SS. 2016. Current applications and different approaches for microbial L-asparaginase production. Brazilian J Microbiol. 47:77–85. https://doi.org/10.1016/j.bjm.2016.10.004
Calderón-Toledo S, Horue M, Alvarez VA, Castro GR, Zavaleta AI. 2021. Isolation and partial characterization of Komagataeibacter sp. SU12 and optimization of bacterial cellulose production using Mangifera indica extracts. J Chem Technol Biotechnol.(June). https://doi.org/10.1002/jctb.6839
Canales PE, Chávez-hidalgo EL, Zavaleta AI. 2014. Caracterización de bacterias halófilas productoras de amilasas aisladas de las Salinas de San Blas en Junín. Rev Colomb Biotecnol. XVI(2). https://doi.org/10.15446/rev.colomb.biote.v16n2.43763
Chakravarty N, Priyanka, Singh J, Singh RP. 2021. A potential type-II L-asparaginase from marine isolate Bacillus australimaris NJB19: Statistical optimization, in silico analysis and structural modeling. Int J Biol Macromol. 174:527–539. https://doi.org/10.1016/j.ijbiomac.2021.01.130
Ciesarovát Z, Kukurová K, Bednáriková A, Marková L, Baxa S. 2009. Improvement of cereal product safety by enzymatic way of acrylamide mitigation. Czech J Food Sci. 27(SPEC. ISS.):1–3. https://doi.org/10.17221/1061-cjfs
Dinarvand B, Rezaei PF, Akbari N, Branch A. 2020. Optimization of L-asparaginase production from a Lactobacillus sp. isolated from traditional dairy products. Biol J Microorg. 9(34):71–86. http://doi.org/10.22108/bjm.2020.121143.1283
Dyall-Smith M, Holmes M, Kamekura M, Lam W, Nuttall S, Bath C, Pfeiffer M, Santos F, Eichler J, Mcalpine T. 2009. The Halohandbook. Dyall-Smith M, editor.
Ebrahiminezhad A, Rasoul-Amini S, Ghasemi Y. 2011. L-Asparaginase production by moderate halophilic bacteria isolated from Maharloo Salt Lake. Indian J Microbiol. 51(3):307–311. https://doi.org/10.1007/s12088-011-0158-6
Ebrahimipour G, Yaghoobi Avini M, Ghorbanmovahhed M. 2020. Isolation and characterization of glutaminase-free L-asparaginase produced by Staphylococcus sp. MGM1. SciMedicine J. 2(2):46–55. https://doi.org/10.28991/scimedj-2020-0202-1
El-Bessoumy AA, Sarhan M, Mansour J. 2004. Production and purification of L-Asparaginase from Pseudomonas aeruginosa 50071 using solid-state fermentation. J Biochem Mol Biol. 37(4):387–393. https://doi.org/10.5483/bmbrep.2004.37.4.387
El-Dein MMN, Mousa MM, Metwally NE. 2019. Isolation and survey of L-asparaginase producing bacteria from soil. J Egypt Acad Soc Environ Dev. 20(1):19–29. https://doi.org/10.21608/jades.2019.67651
Erva RR, Venkateswarulu TC, Pagala B. 2018. Multi level statistical optimization of l-asparaginase from Bacillus subtilis VUVD001. 3 Biotech. 8(1). https://doi.org/10.1007/s13205-017-1020-2
Fonseca MHG, Fiúza TDS, Morais SB, Souza TACB, Trevizani R. 2021. Circumventing the side effects of L-asparaginase. Biomed Pharmacother. 139:111616. https://doi.org/10.1016/j.biopha.2021.111616
Garaev MM, Golub EI. 1977. Mechanism of action of glucose on L-asparaginase synthesis by Escherichia coli bacteria. Mikrobiologiia. 46(3):433–439
Gholamian S, Gholamian S, Nazemi A, Miri Nargesi M. 2013. Optimization of culture media for L-asparaginase production by newly isolated bacteria, Bacillus sp. GH5. Microbiology 82(6):856–863. https://doi.org/10.1134/S0026261714010032
Ghosh S, Murthy S, Govindasamy S, Chandrasekaran M. 2013. Optimization of L-asparaginase production by Serratia marcescens (NCIM 2919) under solid state fermentation using coconut oil cake. Sustain Chem Process. 1(1):1–8. https://doi.org/10.1186/2043-7129-1-9
Gilavand F, Marzban A, Kavyanifard A. 2019. L-Asparaginase-producing Rouxiella species isolation, antileukemia activity evaluation, and enzyme production optimization. Res Mol Med. 6(3):40–59. https://doi.org/10.18502/rmm.v6i3.4608
Golbabaie A, Nouri H, Moghimi H, Khaleghian A. 2020. l-asparaginase production and enhancement by Sarocladium strictum: In vitro evaluation of anti-cancerous properties. J Appl Microbiol. 129(2):356–366. https://doi.org/10.1111/jam.14623
Gulati R, Saxena RK, Gupta R. 1997. A rapid plate assay for screening L-asparaginase producing micro-organisms. Lett Appl Microbiol. 24(1):23–26. https://doi.org/10.1046/j.1472-765X.1997.00331.x
Hall T, Biosciences I, Carlsbad C. 2011. BioEdit: An important software for molecular biology. GERF Bull Biosci. 2:60–61. https://doi.org/10.4236/ajmb.2013.32010
Huang X, Madan A. 1999. CAP3: A DNA sequence assembly program. Genome Research 9(9):868–877. https://doi.org/10.1101/gr.9.9.868
Izadpanah Qeshmi F, Javadpour S, Malekzadeh K, Tamadoni Jahromi S, Rahimzadeh M. 2014. Persian Gulf is a bioresource of potent L-asparaginase producing bacteria: Isolation & molecular differentiating. Int J Environ Res. 8(3):813–818. https://doi.org/10.22059/ijer.2014.774
Jetti J, Jetti A, Perla R. 2017. Production of L-Aparaginase by Using Pectobacterium carotovorum. J Probiotics Heal. 5(01):1–6. https://doi.org/10.4172/2329-8901.1000168
Jha SK, Pasrija D, Singh HR, Nigam VK, Vidyarthi AS. 2014. Batch production and media optimization of anti-leukemic L-asparaginase from Pseudomonas fluorescens by Taguchi DOE methodology. J Appl Pharm Sci. 4(12):085–089. https://doi.org/10.7324/JAPS.2014.41215
Kiranmayi MU, Poda S, Vijayalakshmi M. 2014. Production and optimization of L-asparaginase by an Actinobacterium isolated from Nizampatnam mangrove ecosystem. J Environ Biol. 35(5):799–805.
Kumar S, Stecher G, Li M, Knyaz C, Tamura K. 2018. MEGA X: Molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol. 35(6):1547–1549. https://doi.org/10.1093/molbev/msy096
Le SQ, Gascuel O. 2008. An improved general amino acid replacement matrix. Mol Biol Evol. 25(7):1307–1320. https://doi.org/10.1093/molbev/msn067
Mahajan RV, Saran S, Kameswaran K, Kumar V, Saxena RK. 2012. Efficient production of l-asparaginase from Bacillus licheniformis with low-glutaminase activity: Optimization, scale up and acrylamide degradation studies. Bioresour Technol. 125:11–16. https://doi.org/10.1016/j.biortech.2012.08.086
Menegat FB, Baldo C, Colabone Celligoi MAP, Buzato JB. 2016. Optimization of asparaginase production from Zymomonas mobilis by continuous fermentation. Acta Sci Biol Sci. 38(2):163–168.
Moghadam P, Khadivi Derakhshan F. 2019. Isolation and Identification of Halophilic Bacteria Producing Asparaginase Enzyme from Urmia Lake. Pathobiol Res. 22(4):197–202.
Mohan Kumar NS, Shimray CA, Indrani D, Manonmani HK. 2014. Reduction of acrylamide formation in sweet bread with L-asparaginase treatment. Food Bioprocess Technol. 7(3):741–748. https://doi.org/10.1007/s11947-013-1108-6
Montes J, Canales PE, Flores-Santos JC, Saavedra S, Hurtado-Gómez A, Esquerre-Huallpa C, Zavaleta AI. 2021. Bacteria producing L -asparaginase isolated from Peruvian saline environments. Manglar. 18(2):193–199. https://doi.org/10.17268/manglar.2021.026
Moorthy V, Ramalingam A, Sumantha A, Shankaranaya RT. 2010. Production, purification and characterisation of extracellular L-asparaginase from a soil isolate of Bacillus sp. African J Microbiol Res. 4(18):1862–1867.
Mostafa Y, Alrumman S, Alamri S, Hashem M, Al-izran K, Alfaifi M, Elbehairi SE, Taha T. 2019. Enhanced production of glutaminase-free L-asparaginase by marine Bacillus velezensis and cytotoxic activity against breast cancer cell lines. Electron J Biotechnol. 42:6–15. https://doi.org/10.1016/j.ejbt.2019.10.001
Muslim SN, AL-Kadmy IMS, Hussein NH, Mohammed Ali AN, Dwaish AS. 2016. Extraction and Purification of L-asparaginase produced by Acinetobacter baumannii and their antibiofilm activity against some pathogenic bacteria. Int J Biotechnol. 5(1):7–14. https://doi.org/10.18488/journal.57/2016.5.1/57.1.7.14
Muttucumaru N, Powers SJ, Elmore JS, Dodson A, Briddon A, Mottram DS, Halford NG. 2017. Acrylamide-forming potential of potatoes grown at different locations, and the ratio of free asparagine to reducing sugars at which free asparagine becomes a limiting factor for acrylamide formation. Food Chem. 220:76-86. https://doi.org/10.1016/j.foodchem.2016.09.199
Offman MN, Krol M, Patel N, Krishnan S, Liu J, Saha V, Bates PA. 2011. Rational engineering of L-asparaginase reveals importance of dual activity for cancer cell toxicity. Blood. 117(5):1614-21. https://doi.org/10.1182/blood-2010-07-298422
Pal Roy M, Das V, Patra A. 2019. Isolation, purification and characterization of an extracellular L-asparaginase produced by a newly isolated Bacillus megaterium strain MG1 from the water bodies of Moraghat forest, Jalpaiguri, India. J Gen Appl Microbiol. 65(3):137–144. https://doi.org/10.2323/jgam.2018.07.004
Patel P, Panseriya H, Dave B. 2021. Development of Process and Data Centric Inference System for Enhanced Production of L-Asparaginase From Halotolerant Bacillus licheniformis PPD37. Appl Biochem Biotechnol. https://doi.org/10.21203/rs.3.rs-651645/v1
Patil RC, Jadhav LB. 2017. Screening and optimization of L-asparaginase production from Bacillus species. IOSR J Biotechnol Biochem. 03(03):32–36. https://doi.org/10.9790/264x-03033236
Pradhan B, Dash SK, Sahoo S. 2013. Screening and characterization of extracelluar L-asparaginase producing Bacillus subtilis strain hswx88, isolated from Taptapani hotspring of Odisha, India. Asian Pac J Trop Biomed. 3(12):936–941. https://doi.org/10.1016/S2221-1691(13)60182-3
Prakash P, Singh HR, Jha SK. 2020. Production, purification and kinetic characterization of glutaminase free anti-leukemic L-asparaginase with low endotoxin level from novel soil isolate. Prep Biochem Biotechnol. 50(3):260–271. https://doi.org/10.1080/10826068.2019.1692221
Prihanto AA, Wakayama M. 2014. Combination of environmental stress and localization of l-asparaginase in Arthrospira platensis for production improvement. 3 Biotech. 4(6):647–653. https://doi.org/10.1007/s13205-014-0215-z
Prihanto AA, Yanti I, Murtazam MA, Jatmiko YD. 2019. Optimization of glutaminase-free L-asparaginase production using mangrove endophytic Lysinibacillus fusiformis B27. F1000Research. 8:1938. https://doi.org/10.12688/f1000research.21178.1
Rahimzadeh M, Poodat M, Javadpour S, Qeshmi FI, Shamsipour F. 2016. Purification, Characterization and Comparison between Two New L-asparaginases from PG03 and PG04. Open Biochem J. 10(1):35–45. https://doi.org/10.2174/1874091x01610010035
Rifai L, Saleh FA. 2020. A Review on Acrylamide in Food: Occurrence, Toxicity, and Mitigation Strategies. International Journal of Toxicology 39(2):93-102. https://doi.org/10.1177/1091581820902405
Setiawan R, Larasati DR. 2019. Screening of bacteria producing asparaginase free of glutaminase and urease from hot springs in west Sulawesi. Biosaintifika J Biol Biol Educ. 11(2):218–225. https://doi.org/10.15294/biosaintifika.v11i2.17435
Shah AJ, Karadi RV, Parekh PP. 2010. Isolation, optimization and production of L-asparaginase from coliform bacteria. Asian J Biotechnol. 2(3):169–177. https://doi.org/ 10.3923/ajbkr.2010.169.177
Shaikh Khaleed AN, Khobragade RM. 2016. Screening and Optimization of L-Asparginase Produced by Bacteria Isolated from Soil of Solapur Region. Int J Sci Res. 5(9):231–235.
Shakambari G, Ashokkumar B, Varalakshmi P. 2019. L-asparaginase – A promising biocatalyst for industrial and clinical applications. Biocatal Agric Biotechnol. 17(October 2018):213–224. https://doi.org/10.1016/j.bcab.2018.11.018
Shakambari G, Sumia BM, Ashokkumar B, Palanivelu P, Varalakshmi P. 2020. Industrial effluent as a substrate for glutaminase free L-asparaginase production from Pseudomonas plecoglossicida strain RS1; media optimization, enzyme purification and its characterization. RSC Adv. 10:31205. https://doi.org/10.1039/b000000x
Shifrin S, Parrott CL. 1974. In vitro assembly of L asparaginase subunits. J Biol Chem. 249(13):4175–4180. https://doi.org/10.1016/s0021-9258(19)42499-x
Shirazian P, Asad S, Amoozegar MA. 2016. The potential of halophilic and halotolerant bacteria for the production of antineoplastic enzymes: L-Asparaginase and L-glutaminase. EXCLI J. 15:268–279. https://doi.org/10.17179/excli2016-146
Sudhir AP, Dave BR, Prajapati AS, Panchal K, Patel D, Subramanian RB. 2014. Characterization of a recombinant glutaminase-free L-Asparaginase (ansA3) enzyme with high catalytic activity from Bacillus licheniformis. Appl Biochem Biotechnol. 174(7):2504–2515. https://doi.org/10.1007/s12010-014-1200-z
Venil CK, Nanthakumar K, Karthikeyan K, Lakshmanaperumalsamy P. 2009. Production of L-asparaginase by Serratia marcescens SB08: Optimization by response surface methodology. Iran J Biotechnol. 7(1):10–18.
War Nongkhlaw FM, Joshi SR. 2015. L-Asparaginase and antioxidant activity of endophytic bacteria associated with ethnomedicinal plants. Indian J Biotechnol. 14(1):59–64.
Zenatti PP, Migita NA, Cury NM, Mendes-Silva RA, Gozzo FC, de Campos-Lima PO, Yunes JA, Brandalise SR. 2018. Low Bioavailability and High Immunogenicity of a New Brand of E. coli L-Asparaginase with Active Host Contaminating Proteins. EBioMedicine. 30:158–166. https://doi.org/10.1016/j.ebiom.2018.03.005
Zolfaghar M, Amoozegar MA, Khajeh K, Babavalian H, Tebyanian H. 2019. Isolation and screening of extracellular anticancer enzymes from halophilic and halotolerant bacteria from different saline environments in Iran. Mol Biol Rep. 46(3):3275–3286. https://doi.org/10.1007/s11033-019-04787-7
Zuo S, Zhang T, Jiang B, Mu W. 2015. Reduction of acrylamide level through blanching with treatment by an extremely thermostable L-asparaginase during French fries processing. Extremophiles. 19(4):841–851. https://doi.org/10.1007/s00792-015-0763-0
Downloads
Published
Issue
Section
License
Copyright (c) 2023 Susana Calderón-Toledo, Ysamar Tapia Bañez, Karim Jiménez-Aliaga, Cynthia Esquerre-Huallpa, Amparo Iris Zavaleta
This work is licensed under a Creative Commons Attribution 4.0 International License.
AUTHORS RETAIN THEIR RIGHTS:
a. Authors retain their trade mark rights and patent, and also on any process or procedure described in the article.
b. Authors retain their right to share, copy, distribute, perform and publicly communicate their article (eg, to place their article in an institutional repository or publish it in a book), with an acknowledgment of its initial publication in the Revista Peruana de Biologia.
c. Authors retain theirs right to make a subsequent publication of their work, to use the article or any part thereof (eg a compilation of his papers, lecture notes, thesis, or a book), always indicating its initial publication in the Revista Peruana de Biologia (the originator of the work, journal, volume, number and date).
