Spirulina maxima L-asparaginase: Immobilization, Antiviral and Antiproliferation Activities

Author(s): Hanaa H. Abd El-Baky*, Gamal S. El-Baroty*

Journal Name: Recent Patents on Biotechnology

Volume 14 , Issue 2 , 2020

Become EABM
Become Reviewer

Graphical Abstract:


Background: L-asparaginase (L-AsnA) enzyme has gained significant attention in the food, biocatalysts and pharmaceutics industry. It (L-AsnA) has been widely used in food processing industries as a promising acrylamide mitigating agent and as a therapeutic agent in the treatment of certain human cancers.

Objective: Based on US Patent (4,433,054; 1984), L-asparaginase (L-AsnA) enzyme is immobilized by admixing the active enzyme on the polysaccharide to be in a gel form. The storage stability of immobilized L-AsnA enzyme and its anti-proliferation and antiviral activity were determined.

Methods: In the present study, S. maxima was cultured at large scales (300 liter) for the production of enough extracellular L-asparaginase (L-AsnA) using modified (high N concentration) Zarrouk medium as we reported in a previous study. L-AsnA was immobilized on natural polymers, as agar cake beads, agarose pieces and gelatin blocks, in order to evaluate the efficiency of physical entrapment techniques. Anti-proliferation properties of L-AsnA against lung carcinoma A549, hepatocellular carcinoma Hep-G2 and prostate carcinoma PC3 human cancer cell lines were assessed by the MTT cell viability method. In addition, the antiviral activity against Coxsackie B3 (CSB3) Virus was assessed.

Results: The highest L-AsnA immobilized activity and immobilization yield were achieved with agar cakes bead. The purified S. maxima L-AsnA showed good antiviral activity against Coxsackie B3 (CSB3) Virus in a dose-dependent manner with an IC50 value 17.03 μg/ml. The antiviral mode of action is presumably due to their capability of inhibiting attachment, blocking the adsorption and penetration event of the viral replication cycle with 89.24%, 72.78% and 72.78%, respectively. Also, S. maxima L-AsnA showed anti-proliferation effect against lung carcinoma A549, hepatocellular carcinoma Hep-G2 and prostate carcinoma PC3 human cancer cell lines, with an IC50 of 22.54, 24.65 and 56.61 μg/ml, respectively.

Conclusion: It is interesting to favor L-asparaginase of S. maxima which showed antiviral activity and anti-proliferation effect against different types of human cell lines. Thus, S. maxima microalgae might be a good source for L-AsnA enzymes and can be immobilized on natural polymers.

Keywords: L-asparaginase, anti-proliferation activity, antiviral activity, Spirulina maxima, enzyme, virus.

Chibata I, Tosa T, Takata I. Enzymatic active substance immobilized in a polysaccharide gel matrix. US4433054, 1984.
Zarrouk C. Contribution a letuded unecyanobacterie: influence de divers facteurs physiques et chimiquessur la croissance et la photosynthese de Spirulina maxima (Setchell et Gardner) Geitler. University of Paris, Paris 1966. [PhD thesis]
Abd El Baky HH, El Baroty GS. Optimization of growth conditions for purification and production of L-asparaginase by Spirulina maxima. Evid Based Complement Alternat Med 2016; 20161785938
Monajati M, Borandeh S, Hesami A, Mansouri D, Tamaddon AM. Immobilization of L-asparaginase on aspartic acid functionalized grapheme oxide nanosheet: enzyme kinetics and stability studies. Chem Eng J 2018; 354: 1153-63.
Kamble VP, Rao RS, Borkar PS, Khobragade CN, Dawane BS. Purification of L-asparaginase from a bacteria Erwinia carotovora and effect of a dihydropyrimidine derivative on some of its kinetic parameters. Indian J Biochem Biophys 2006; 43(6): 391-4.
[PMID: 17285805]
Shi R, Liu Y, Mu Q, Jiang Z, Yang S. Biochemical characterization of a novel L-asparaginase from Paenibacillus barengoltzii being suitable for acrylamide reduction in potato chips and mooncakes. Int J Biol Macromol 2017; 96: 93-9.
[http://dx.doi.org/10.1016/j.ijbiomac.2016.11.115] [PMID: 27919811]
Arjun JK, Aneesh B, Kavitha T, Krishnan KH. Therapeutic L-asparaginase activity of bacteria isolated from marine sediments. Int J Pharma Sci 2016; 8(4): 229-34.
Jayachandran D, Chityala S, Prabhu AA, Dasu VV. Cationic reverse micellar based purification of recombinant glutaminase free L-asparaginase II of Bacillus subtilis WB800N from fermentation media. Protein Expr Purif 2019; 157: 1-8.
[http://dx.doi.org/10.1016/j.pep.2019.01.002] [PMID: 30615939]
Sun Z, Qin R, Li D, et al. A novel bacterial type II l-asparaginase and evaluation of its enzymatic acrylamide reduction in French fries. Int J Biol Macromol 2016; 92: 232-9.
[http://dx.doi.org/10.1016/j.ijbiomac.2016.07.031] [PMID: 27402458]
Kuma NS, Manonmani HK. Purification, characterization and kinetic properties of extracellular L-asparaginase produced by Cladosporium sp. World J Microbiol Biotechnol 2013; 29(4): 577-87.https://link.springer.com/article/10.1007%2Fs11274-012-1213-0
Izadpanah QF, Homaei A, Fernandes P, Javadpour S. Marine microbial L-asparaginase: Biochemistry, molecular approaches and applications in tumor therapy and in food industry. Microbiol Res 2018; 208: 99-112.
[http://dx.doi.org/10.1016/j.micres.2018.01.011] [PMID: 29551216]
Darvishi F, Faraji N, Shamsi F. Production and structural modeling of a novel asparaginase in Yarrowia lipolytica. Int J Biol Macromol 2019; 125: 955-61.
[http://dx.doi.org/10.1016/j.ijbiomac.2018.12.162] [PMID: 30576739]
Ebrahiminezhad A, Rasoul-Amini S, Ghoshoon MB, Ghasemi Y. Chlorella vulgaris, a novel microalgal source for L-asparaginase production. Biocatal Agric Biotechnol 2014; 3: 214-7.
Prihanto AA, Wakayama M. Combination of environmental stress and localization of L-asparaginase in Arthrospira platensis for production improvement. 3 Biotech. 2014; 4(6): 647-53.
[http://dx.doi.org/10.1007/s13205-014-0215-z] [PMID: 28324309]
Kumar S, Yadav RK, Negi S. A comparative study of immobilized lipase produced from Penicillium chrysogenum SNP5 on two deferent anionic carriers for its pH and thermo stability. Indian J Biotechnol 2014; 13(3): 301.https://pdfs.semanticscholar.org/d9af/90e03e5f02218a7698eb957e43123dad5880.pdf
Garcia-Galan CÁ, Berenguer-Murcia R, Fernandez-Lafuente RC. Rodrigues. Potential of deferent enzyme immobilization strategies to improve enzyme performance. Adv Synth Catal 2011; 353: 2885-904.
Barbosa O, Torres R, Ortiz C, Berenguer-Murcia A, Rodrigues RC, Fernandez-Lafuente R. Heterofunctional supports in enzyme immobilization: from traditional immobilization protocols to opportunities in tuning enzyme properties. Biomacromolecules 2013; 14(8): 2433-62.
[http://dx.doi.org/10.1021/bm400762h] [PMID: 23822160]
Goncalves MC, Kieckbusch TG, Perna RF, Fujimoto JT, Morales SAV, Romanelli JP. Trends on enzyme immobilization researches based on bibliometric analysis. Process Biochem 2019; 76: 95-110.
Bedade DK, Sutar YB, Singhal RS. Chitosan coated calcium alginate beads for covalent immobilization of acrylamidase: Process parameters and removal of acrylamide from coffee. Food Chem 2019; 275: 95-104.
[http://dx.doi.org/10.1016/j.foodchem.2018.09.090] [PMID: 30724265]
Bahraman F, Alemzadeh I. Optimization of L-asparaginase immobilization onto calcium alginate Beads. Chem Eng Commun 2017; 204: 216-20.
Afify AM, El Baroty SG, El Baz FK, Abd El Baky HH, Murad S. Antioxidant activity of protein hydrolysates derived from blue-green algae Spirulina platensis extracted with three different methods and treated with enzymes. Bioscience Research 2017; 14(3): 485-97. https://scholar.cu.edu.eg/?q=elbarotys/files/22_biomass_spirulina_protein.pdf
Bhat M, Marar T. Cytotoxic effect of purified Lasparaginase from Salinicoccus sp. M KJ997975. Int J CurrMicrobiol App Sci 2015; 4(4): 701-12. https://www.ijcmas.com/vol-4-4/Manish%20Bhat%20and%20Thankamani%20%20Marar.pdf
Strbánová-Necinová S, Svobodová O, Vránová J. Localization of L-asparaginase in Escherichia coli. Folia Microbiol (Praha) 1974; 19(6): 474-8.https://link.springer.com/article/10.1007/BF02872912
[http://dx.doi.org/10.1007/BF02872912] [PMID: 4611872]
Mahajan R, Gupta VK, Sharma J. Comparison and suitability of gel matrix for entrapping higher content of enzymes for commercial applications. Indian J Pharm Sci 2010; 72(2): 223-8.
[http://dx.doi.org/10.4103/0250-474X.65010]] [PMID: 20838527]
Prakash O, Puliga S, Upadhyay LSB. Immobilization of watermelon (Citrus vulgaris) urease in agarose gel for urea estimation. Biotech Biopro Eng 2007; 12: 1315. https://link.springer.com/article/10.1007/BF03028638
Gulati R, Saxena RK, Gupta R. A rapid plate assay for screening L-asparaginase producing micro-organisms. Lett Appl Microbiol 1997; 24(1): 23-6.
Xiong HR, Shen YY, Lu L, et al. The inhibitory effect of Rheum palmatum against coxsackievirus B3 in vitro and in vivo. Am J Chin Med 2012; 40(4): 801-12.
[http://dx.doi.org/10.1142/S0192415X12500607] [PMID: 22809033]
Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 1983; 65(1-2): 55-63.
[http://dx.doi.org/10.1016/0022-1759(83)90303-4] [PMID: 6606682]
Hansen J, Lacis A, Prather M. Greenhouse effect of chlorofluorocarbons and other trace gases. J Geophys Res 1989; 94: 16417-21.
Vala AK, Sachaniya B, Dudhagara D, et al. Characterization of L-asparaginase from marine-derived Aspergillus niger AKV-MKBU, its antiproliferative activity and bench scale production using industrial waste. Int J Biol Macromol 2018; 108: 41-6.
[http://dx.doi.org/10.1016/j.ijbiomac.2017.11.114] [PMID: 29175524]
Shakambari G, Ashokkumar B, Varalakshmi P. L-asparaginase - A promising biocatalyst for industrial and clinical applications. Biocatal Agric Biotechnol 2019; 17: 213-22433.
Pritsa AA, Papazisis KT, Kortsaris AH, Geromichalos GD, Kyriakidis D. Antitumor activity of L-asparaginase from Thermus thermophilus. Anticancer Drugs 2001; 12(2): 137-42.
[http://dx.doi.org/10.1097/00001813-200102000-00007]] [PMID: 11261887]
Abakumova OIu, Podobed OV, Borisova AA, et al. [Antitumor activity of L-asparaginase from Yersinia pseudotuberculosis]. Biomed Khim 2008; 54(6): 712-9.https://www.ncbi.nlm.nih.gov/pubmed/19205431
[PMID: 19205431]
Pritsa AA, Kyriakidis DA. L-asparaginase of Thermus thermophilus: purification, properties and identification of essential amino acids for its catalytic activity. Mol Cell Biochem 2001; 216(1-2): 93-101.https://link.springer.com/article/10.1023/A:1011066129771
[http://dx.doi.org/10.1023/A:1011066129771] [PMID: 11216870]
Loureiro CB, Borges KS, Andrade AF, Tone LG, Said S. Purification and biochemical characterization of native and pegylated Form of L-asparaginase from Aspergillus terreus and evaluation of its antiproliferative activity. Adv Microbiol 2012; 2: 138-45.
Selvam K, Vishnupriya B. Industrial applications of newly isolated marine actinomycetes Streptomyces spp. J Biosci Res 2013; 4(2): 58-65.
Rani SA. Lalithasundaram, Praveesh Bahuleyan Vasantha. Vitro anti-oxidant and anti-cancer activity of L-Asparaginase from Aspergillus flavus (KUFS20). Asian J Pharm ClinRes 2011; 4(2): 174-7. https://innovareacademics.in/journal/ajpcr/Vol4Suppl2/682.pdf
Hau AM, Greenwood JA, Löhr CVJD, et al. Coibamide A induces mTOR-independent autophagy and cell death in human glioblastoma cells. PLoS One 2013; 8(6)e65250
[http://dx.doi.org/10.1371/journal.pone.0065250] [PMID: 23762328]
Abd El Baky HH, El-Baroty GS. The potential use of microalgae carotenoids as dietary supplements and natural preservative ingredients. J Aquat Food Prod Technol 2017; 22(4): 392-406.
Sin J, Mangale V, Thienphrapa W, Gottlieb RA, Feuer R. Recent progress in understanding coxsackievirus replication, dissemination, and pathogenesis. Virology 2015; 484: 288-304.
[http://dx.doi.org/10.1016/j.virol.2015.06.006] [PMID: 26142496]
Cappel R, Klastersky J, Thiry L. Anti-herpetic activity of a combination of L-asparaginase and cytosine arabinoside used in vitro. Postgrad Med J 1973; 49: 428.

Rights & PermissionsPrintExport Cite as

Article Details

Year: 2020
Page: [154 - 163]
Pages: 10
DOI: 10.2174/1872208313666191114151344
Price: $65

Article Metrics

PDF: 11