Screening and Characterization of L-asparaginase Producing Bacterial Strains from the Soil in Bangladesh
Journal of Advances in Microbiology,
Although the enzyme L-asparaginase (L-ASNase) from Escherichia coli, Erwinia and Serratia has been applied to treat certain lymphomas and leukemias, several medical complications such as severe immunological responses leading to hypersensitivity, anaphylaxis, etc. have limited its application. The researchers have documented that such impediments are due to the different biochemical and kinetic properties of L-ASNase, which are directly dependent on genetic variations in microbial strains. Thus, there is a compelling need to explore novel L-ASNase producing microorganisms that would exhibit different serological properties while retaining similar and/or better therapeutic effects against cancer cells. Heretofore, L-ASNase producing bacterial strains from Bangladesh have never been isolated and characterized. Therefore, the main objective of this research was to isolate and characterize these strains from unexplored and ecologically different habitats that could lead to developing a potential therapeutic drug with fewer immunological responses and side effects over the existing drugs in order to treat cancer patients in the near future. Two L-ASNase producing bacterial strains were successfully isolated from the soil of Hatirjheel lake in Dhaka for the first time. Molecular characterization revealed that both strains belonged to Pseudomonas aeruginosa and their DNA sequences were submitted to NCBI GenBank. The accession number OK446669 was obtained for the strain of P. aeruginosa EWUKR-1 and OL307081 for P. aeruginosa EWUKR-2. The specific activity of L-ASNase from EWUKR-2 (212.1 ± 14.8 U/mg protein) was significantly higher than that of EWUKR-1 (16.3 ± 0.8 U/mg protein) when they were grown in modified M9 media containing 0.5 g/l glucose at 370C for 24 hours. The experimental results revealed that both of these bacterial strains were extracellular L-ASNase producers. The enzyme from P. aeruginosa EWUKR-2 was partially purified using saturated ammonium sulfate followed by dialysis and concentrated using Vivaspin-20 centrifugal concentrator having MWCO of 30 kDa. The optimum temperature and pH of the partially purified enzyme were 370C and 7.5, respectively. The purification-fold after ammonium sulfate precipitation and yield of the concentrated enzyme were 2.8 and 101%, respectively. SDS-PAGE analysis revealed that the molecular weight of L-ASNase from P. aeruginosa EWUKR-2 was around 43 kDa.
- therapeutic drugs
How to Cite
Yellin TO, Wriston JC. Antagonism of purified asparaginase from guinea pig serum toward lymphoma. Science. 1966;151(3713):998-9.
Broome JD. Evidence that the L-asparaginase activity of guinea pig serum is responsible for its antilymphoma effects. Nature. 1961;191(4793):1114-5.
Abbas AA, Sabbah MA, Kathum OA. Partial purification and cytotoxic activity of L-asparaginase isolated from Escherichia coli. Iraq J Sci. 2010;51:290-4.
Gulati R, Saxena RK, Gupta RA. Rapid plate assay for screening L-asparaginase producing microorganisms [lett]. Appl Microbiol. 1997;24(1):23-6.
Yunis AA, Arimures GK, Russin DJ. Human pancreatic carcinoma (MIA PaCa-2) in continues culture sensitivity to L-asparaginase. Int J Cancer. 1977;19: 218-35.
Biagiotti S, Paoletti MF, Fraternale A, Rossi L, Magnani M. Drug delivery by red blood cells. IUBMB Life. 2011;63(8): 621-31.
Pieters R, Hunger SP, Boos J, Rizzari C, Silverman L, Baruchel A et al. L-asparaginase treatment in acute lymphoblastic leukemia: a focus on Erwinia asparaginase. Cancer. 2011;117(2): 238-49.
Teodor E, Litescu SC, Lazar V, Somoghi R. Hydrogel-magnetic nanoparticles with immobilized L-asparaginase for biomedical applications. J Mater Sci Mater Med. 2009;20(6):1307-14
Kumar S, Dasu VV, Pakshirajan K. Localization and production of novel L-asparaginase from Pectobacterium carotovorum MTCC 1428. Process Biochem. 2010;45(2):223-9.
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.
Agarwal A, Kumar S, Veeranki VD. Effect of chemical and physical parameters on the production of L-asparaginase from a newly isolated Serratia marcescens SK-07 [lett]. Lett Appl Microbiol. 2011;52(4):307-13.
Amena S, Vishalakshi N, Prabhakar M, Dayanand A, Lingappa K. Production, purification and characterization of L-asparaginase from Streptomyces gulbargensis. Braz J Microbiol. 2010;41(1):173-78.
El-Bessoumy AA, Sarhan M, Mansour J. Production, isolation, and purification of L-asparaginase from Pseudomonas aeruginosa 50071 using solid-state fermentation. J Biochem Mol Biol. 2004;37(4):387-93.
Komathi S, Rajalakshmi GR, Savetha S, Balaji S. Isolation, production and partial purification of l-asparaginase from Pseudomonas aeruginosa by solid state fermentation. Sch. Acad. J Pharmacol. 2013;2:55-9.
Izadpanah Qeshmi F, Homaei A, Khajeh K, Kamrani E, Fernandes P. Production of a novel marine Pseudomonas aeruginosa recombinant L-asparaginase: insight on the structure and biochemical characterization. Mar Biotechnol. 2022;24(3):599-613.
Bansal S, Gnaneswari D, Mishra P, Kundu B. Structural stability and functional analysis of L-asparaginase from Pyrococcus furiosus. Biochemistry (Mosc). 2010;75(3):375-81.
Eden OB, Shaw MP, Lilleyman JS, Richards S. Non-randomized study comparing toxicity of Escherichia coli and Erwinia asparaginase in children with leukaemia. Med Pediatr Oncol. 1990;18(6):497-502.
Prescott LM, Harley JP, Klein DA. Procaryotic cell structure and function: introduction to microbiology. 4th ed. New York: McGraw-Hill; 1999. p. 37-72.
Imada A, Igarasi S, Nakahama K, Isono M. Asparaginase and glutaminase activities of microorganisms. J Gen Microbiol. 1973;76(1):85-99.
Distasio JA, Niederman RA, Kafkewitz D, Goodman D. Purification and characterization of L-asparaginase with antilymphoma activity from Vibroi succinogenes. J Biol Chem. 1976;251(22):6929-33.
Kielkopf CL, Bauer W, Urbatsch IL. Bradford assay for determining protein concentration. Cold Spring Harb Protoc. 2020;2020(4):102269.
Sambrook J, Russell D. Molecular cloning: A laboratory manual. Cold Spring Harbor, NY; 2001.
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. 2013;89: 15-23.
Higgins DG, Sharp PM. Clustal: a package for performing multiple sequence alignment on a computer. Gene. 1988;73(1):237-44.
Tamura K, Stecher G, Kumar S. MEGA11: Molecular Evolutionary Genetics Analysis Version 11. Mol Biol Evol. Version 11. 2021;38(7):3022-7.
Holt JG, Noel RK. Bergey's manual of determinative bacteriology. 9th ed. Baltimore: Williams & Wilkins Co.; 1994.
El-Bessoumy AA, Sarhan M, Mansour J. Production, isolation and purification of L-asparaginase from Pseudomonas aeruginosa 50071 using solid state fermen-tation. J Biochem Mol Biol. 2004;37(4):387-93.
Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227(5259):680-5.
Selvakumar N, Chandramohan D, Natrajan R. L-asparaginase activity in marine sediments. Curr Sci. 1977;46:287-90.
Lubkowski J, Palm GJ, Gilliland GL, Derst C, Röhm KH, Wlodawer A. Crystal structure and amino acid sequence of Wolinella succinogenes L-asparaginase. Eur J Biochem. 1996;241(1):201-7.
Bansal S, Srivastava A, Mukherjee G, Pandey R, Verma AK, Mishra P et al. Hyperthermophilic asparaginase mutants with enhanced substrate affinity and antineoplastic activity: structural insights on their mechanism of action. FASEB J. 2012;26(3):1161-71.
Ghosh S, Chaganti SR, Prakasham RS. Polyaniline nanofibers as a novel immobilization matrix for the anti-leukaemia enzyme L-asparaginase. J Mol Cat B Enzym. 2011;74:132-7.
Arrivukkarasan S, Muthusivaramapandian M, Aravindan R, Viruthagiri T. Effect of medium composition and kinetic studies on extracellular and intracellular production of L-asparaginase from Pectobacterium carotovorum. Food Sci Technol Int. 2010;16(2):115-25.
Kodchakorn L, Saisamorn L, Sutheera T, Pairote W, Uraporn S. L-asparaginase production by Bipolaris sp. BR438 isolated from brown rice in Thailand. Chiang Mai J Sci. 2012;37:160-4.
Abstract View: 157 times
PDF Download: 42 times