SCREENING AND PRODUCTION OF ANTICARCINOGENIC ENZYME FROM ESCHERICHIA COLI CTLS20: L - ASPARAGINASE
Abstract
Objective: The objective of this study was attempted to screen the production of L-asparaginase from bacteria isolated from soil samples and its enzymatic activity.
Methods: Screening of L-asparaginase was performed using phenol red indicator growth medium from which the positive strains were chosen based on the colour change. The enzyme production of L-asparaginase was established by submerged fermentation followed by the molecular detection of the efficient bacterial strains.
Results: The enzyme production was undertaken by submerged fermentation with the evaluation of enzymatic activity and protein content. This revealed that the strain Escherichia coli CTLS20 produced a higher yield of L-asparaginase (30.22 IU/mg), 16.91 µg/ml of protein with the specific activity of 1.787 IU/mg when compared with other bacterial strains. The efficient bacterial strains were also confirmed by 16S rRNA sequence as Escherichia coli, Acinetobacter baumnnii, Klebsiella pneumoniae and the phylogenetic tree construction revealed the evolutionary relationship of the bacterial strains.
Conclusion: This study indicated that the bacterial strain E. coli CTLS20 had the ability for the higher production of L-asparaginase. This novel higher yielding bacterial asparaginase is highly desirable as better alternatives in cancer therapy.
Keywords: Soil, L-asparaginase, Submerged fermentation, E. coli, Phylogenetic tree
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References
Warangkar SC, Khobragade CN. Screening, enrichment and media optimization for L–asparaginase production. J Cell Tissue Res 2009;9 Suppl 3:1963-8.
Mohini Patil P, Ravindra Patil H, Vijay Maheshwari L. A novel and sensitive agar plug assay for screening of asparaginase-producing endophytic fungi from Aeglemarmelos. Acta Biol (Szeged) 2012;56 Suppl 2:175-7.
Stecher AL, Morgantetti P, De Deus, Polikarpov I, AbrahaoNeto J. Stability of L-asparaginase-an enzyme used in leukemia treatment. Pharm Acta Helv 1999;74:1-9.
Verma NK, Kumar G, Kaur, Anand S. L-asparaginase: a promising chemotherapeutic agent. Crit Rev Biotechnol 2007;27:45-62.
Devarai Santhoskumar, Kota Sobha. L-asparginase from microbes-a comprehensive review. Adv Bio Res 2012;3 Suppl 4:137-57.
Mash Burn LT, Wriston JC. Tumor inhibitory effect of L-asparaginase from E. coli. Arch Biochem Biophys 1964;105:450-2.
Stern ML, Phillips AW, Gottlieb AJ. Physical properties of L-asparaginase from Serratia marcescens. J Bacteriol 1976;125:719-27.
Mesas JM, Gil JA, Martin JF. Characterization and partial purification of L-asparaginase from Corynebacterium glutamicum. J Gen Microbiol 1990;136:515-9.
Muley RG, Sarker S, Ambedkar S, Nail SR. Influence of alkali-treated corn steep liquor containing medium on protein production by Staphylococcus aureus. Folia Microbiol 1998;43:31-4.
Aghaiypour K, Wlodawer A, Lubkowski J. Structural basis for the activity and substrate specificity of Erwinia chrysanthemi L-asparaginase. Biochemistry 2001;40:5655-64.
Fisher SH, Wray LV. Bacillus subtilis168 contains two differentially regulated genes encoding L-asparaginase. J Bacteriol 2002;184:2148-54.
Ashraf El-Bessoumy A, Mohamed Sarhan, Jehan Mansour. Production, isolation and purification of L-asparaginase from Pseudomonas aeruginosa 50071 using solid state fermentation. J Biochem Mol Biol 2004;37:387-93.
Drainas C, Pateman JA. L-Asparaginase activity in the fungus Aspergillus nidulans. Biochem Soc Trans 1977;5:259-61.
Sarquis MI, Oliveira EM, Santos AS, Costa GL. Production of L-asparaginase by filamentous fungi. Mem Inst Oswaldo Cruz 2004;99:489-92.
Debajit Borah, Yadav RNS, Ankush Sangra, Lubana Shahin, Anand Kumar Chaubey. Production, purification and process optimization of asparaginase (an anticancer enzyme) from E. coli isolated from sewage water. Asian J Pharm Clin Res 2012;5:202-4.
Mashburn LT, Wriston TJC. Tumor inhibitory effect of L-asparaginase from Escherichia coli. Archives Biochem Biophy 1964;105:451-2.
Sreepad Jois, Shilpa Chandru, Ashajyothi Chavan, Ramachandra Yarapa Lakshmikantha, Padmalatha Rai Satwadi. Study on production, purification, and characterisation of L-asparaginase from Escherichia coli and Pseudomonas aeruginosa. Int J Pharm Chem Biol Sci 2013;3 Suppl 3:565-70.
Saleem Basha N, 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. Trop J Pharm Res 2009;8 Suppl 4:353-60.
Rati Kumari Sinha, Hare Ram Singh, Santosh Kumar Jha. Production, purification and kinetic characterization of L-asparaginase from Pseudomonas fluorescens. Int J Pharm Sci 2015;7:135-8.
Ramraj Upadhyay, Akanksha Saxena, Naveen Kango. Screening and production of tumor inhibitory L-Asparaginase by bacteria isolated from soil. Asian J Pharm Clin Res 2012;5 Suppl 3:135-7.
Dubey RC, Maheshwari DK. Practical microbiology. S Chand Limited. India; 2002. p. 286-91.
John Holt G. Bergey’s manual of determinative bacteriology. 9th Edition. United States of America; 2000.
Younes Ghasemi, Alireza Ebrahiminezhad, Sara Rasoul-Amini, Gholamreza Zarrini, Mohammad Bagher Ghoshoon, Mohammad Javad Raee, et al. An optimized medium for the screening of L-asparaginase production by Escherichia coli. Am J Biochem Biotechnol 2008;4 Suppl 4:422-4.
Heinemann B, Howard AJ. Production of tumor-inhibitory L-asparaginase by submerged growth of Serratia marcescens. Appl Microbiol 1969;18:550-4.
Levenberg B, Hayaishi O. A bacterial pterin deaminase. J Biol Chem 1959;234:955-61.
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with folin phenol reagent. J Biol Chem 1951;193:265-75.
Marmur J. A procedure for the isolation of deoxyribonucleic acid from micro-organisms. J Mol Biol 1961;3 Suppl 2:208.
Richa Jain, Zaidi KU, Yogita Verma, Pooja Saxena. L-asparaginase: a promising enzyme for the treatment of acute lymphoblastic leukaemia. Peoples J Sci Res 2012;5 Suppl 1:29-35.
Kumar Jha S, Divya Pasrija, Rati Kumari Sinha, Hare Ram Singh, Vinod Kumar Nigam, Ambrish Sharan Vidyarthi. Microbial L-asparaginase: a review on current scenario and future prospects. Int J Pharma Sci Res 2012;3 Suppl 9:3076-90.
Kamble KD, Bidwe PR, Muley VY, Kamble LH, Bhadange DG, Musaddiq M. Characterization of L-Asparaginase producing bacteria from water, farm and saline soil. J Biosci Dis 2012;3 Suppl 1:116-9.
Biswaprakash Pradhan, Sashi Dash K, Sabuj Sahoo. Screening and characterization of extracellular L-asparaginase producing Bacillus subtilis strain hswx88, isolated from taptapanihotspring of odisha, India. Asian Pac J Trop Biomed 2013;3:936-41.
Imada A, Igarasi S, Nakahama K, Isono M. Asparaginase and glutaminase activities of microorganisms. J Gen Microbiol 1973;76:85-99.
Ahyar Ahmad, Abdul Muis Patta, Hasnah Natsir. Purification and immobilization of L-asparaginase enzyme from the thermophilic bacteria Bacillus licheniformis strain HSA3-1a. Int J Pharm Biol Sci 2013;4 Suppl 4:274-80.
Cedar H, Schwartz JH. Production of L-asparaginase II by Escherichia coli. J Bacteriol 1968;96:2043-8.
Barnes WR, Vela GR, Dorn GL. Physiology of L-asparaginase synthesis in recombinants of Escherichia coli A-1. Appl Environ Microbiol 1978;35:766-70.
Swain AL, Jasklski M, Houssett D, Mohana Rao JK, Alexander W. Crystal structure of Escherichia coli L-asparaginase, an enzyme used in cancer therapy. Proc Natl Acad Sci USA 1993;90:1474-8.
Richa Jain, Zaidi KU, Yogita Verma, Pooja Saxena. L-Asparaginase: a promising enzyme for the treatment of acute lymphoblastic leukaemia. Peoples J Sci Res 2012;5 Suppl 1:29-35.
Muharram MM, Abulhamd AT, Mounir M, Salem Bekhet. Recombinant expression, purification of L-Asparaginase-II from thermotolerant E. coli strain and evaluation of its antiproliferative activity. Afr J Microbiol Res 2014;8 Suppl 15:1610-9.
Niharika Yadav, Supriya Sarkar. Production of L-asparaginase by Fusarium oxysporum using submerged fermentation. Int J Pharm Sci Invent 2014;3 Suppl 6:32-40.
Sarita Devi, Aditya Kulshreshtha, Ashutosh Kumar Rai, Wamik Azmi. Bench-scale production of L-asparaginase from Erwinia carotovora in a laboratory fermenter. Int J Life Sci Pharma Res 2012;2 Suppl 3:25-35.