PURIFICATION AND CHARACTERIZATION OF L-ASPARAGINASE BY PSEUDONOCARDIA ENDOPHYTICA VUK-10 ISOLATED FROM NIZAMPATNAM MANGROVE ECOSYSTEM
Objective: L-asparaginase has been a promising therapeutic agent in the treatment of acute lymphoblastic leukaemia. In the present study a rare actino bacterial strain Pseudonocardia endophytica VUK-10 isolated from Nizampatnam mangrove ecosystem was explored for the production of L-asparaginase.
Methods: The extracellular L-asparaginase enzyme was purified to homogeneity from the P. endophytica VUK-10 strain. The crude culture filtrate was subjected to different purification steps including ammonium sulphate fractionation followed by separation on Sephadex G-100 gel filtration and CM-Sephadex C-50 ion exchange chromatography to obtain a pure enzyme preparation.
Results: The enzyme was purified 96 fold and showed a final specific activity of 702.04 IU/mg with a 61% yield. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified enzyme revealed its nature as single peptide chain with molecular weight of 120 kDa. This is the first report on production and purification of L-asparaginase from P. endophytica of mangrove origin.
Conclusion: The extracellular L-asparaginase of the P. endophytica may be effectively used as potential chemotherapeutic agent.
Keywords: Mangrove ecosystem, Pseudonocardia endophytica, L-asparaginase, Purification, SDS-PAGE
Amy IS, Mariusz J, Dominique H, Mohana Rao JK. Crystal structure of Escherichia coli L-asparaginase, an enzyme used in cancer therapy. Proc Natl Acad Sci USA 1993;90:1474-8.
Surajit D, Lyla PS, Ajmal Khan S. Marine microbial diversity and ecology: importance and future perspectives. Curr Sci 2006;90:1325-35.
Clementi A. La desamidation enzymatic del asparagine chez les differentes especes animals et la signification physiologique de sa presence dans organisms. Arch Int Physiol 1922;19:369.
Kidd JG. Regression of transplanted lymphomas induced in vivo by means of normal guinea pig serum. The course of transplanted cancers of various kinds in mice and rats given guinea pig serum, horse serum or rabbit serum. J Exp Med 1953;98:565-82.
Mashburn LT, Wriston JC Jr. Tumor inhibitory effect of L-asparaginase from Escherichia coli. Arch Biochem Biophys 1964;105:450-2.
Peterson RE, Ciegler A. L-asparaginase production by Erwinia aroideae. Appl Microbiol 1969;18:64-7.
Lee SM, Wroble MH, Ross JT. L-asparaginase from Erwinia carotovora-an improved recovery and purification process using affinity chromatography. Appl Biochem Biotechnol 1989;22:1-11.
Cedar H, Schwartz JH. Production of L-Asparaginase II by Escherichia coli. J Bacteriol 1968;96:2043-8.
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.
Wriston JC Jr. Asparaginase. Methods Enzymol 1985;113:608-18.
Maladkar NK, Singh VK, Naik SR. Fermentative production and isolation of L-asparaginase from Erwinia carotovora, EC-113. Hind Antibiot Bull 1993;35:77-86.
Theantana T, Hyde KD, Lumyong S. Asparaginase production by endophytic fungi from Thai medicinal plants: cytotoxicity properties. Int J Integr Biol 2009;7:1-8.
Dhanam Jayam G, Kannan S. L-asparaginase-Types, perspectives and applications. Adv Biotechnol 2013;13:1-5.
Verma N, Kumar K, Kaur G, Anand S. L-Asparaginase: a promising chemotherapeutic agent. Crit Rev Biotechnol 2007;27:45-62.
McCredie JA, Inch WR, Kruuv J, Watson TA. The rate of tumor growth in animals. Growth 1965;29:331-47.
Das S, Lyla PS, Khan SA. Marine microbial diversity and ecology: importance and future perspectives. Curr Sci 2006;90:25-9.
Dhevagi P, Poorani E. Isolation and characterization of L-asparaginase from marine actinomycetes. Indian J Biotechnol 2006;5:514-20.
Saleem Basha N, Rekha R, Komala M, Ruby S. Production of extracellular anti-leukemic enzyme L-asparaginase from marine actinomycetes by solid state and submerged fermentation: purification and characterization. Trop J Pharm Res 2008;8:353-60.
Pokrovskaya MV, Aleksandrova SS, Pokrovsky VS, Omeljanjuk NM, Borisova AA, Anisimova NY, et al. Cloning, expression and characterization of the recombinant Yersinia Pseudotuberculosis L-asparaginase. Protein Expression Purif 2012;82:150-4.
Sivasankar P, Sugesh S, Vijayanand P, Sivakumar K, Vijayalakshmi S, Balasubramanian T, et al. Efficient production of L-asparaginase by marine Streptomyces sp. isolated from bay of Bengal, India. Afr J Microbiol Res 2013;7:4015-21.
Usha Kiranmayi M, Vijayalakshmi M, Sudhakar P, Sreenivasulu K. Isolation, Identification and molecular characterization of rare actinomycetes from mangrove ecosystem of nizampatnam. Malaysian J Microbiol 2012;8:83-91.
Imada A, Igarasi S, Nakahama K, Isono M. Asparaginase and glutaminase activities of microorganisms. J Gen Microbiol 1973;76:85-9.
Usha Kiranmayi M, Sudhakar P, Vijayalakshmi M. Production and optimization of L-Asparaginase by an actinobacterium isolated from nizampatnam mangrove ecosystem. J Environ Biol 2014;35:799-805.
Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227:680-5.
Bhat MR, Marar T. Purification and characterization of L-asparaginase from Salinicoccus spp. M KJ997975. Int J Curr Trends Sci Technol 2015;14:42-8.
Amena S, Vishalakshi N, Prabhakar M, Dayananand A, Lingappa K. Production, purification and characterization of L-asparaginase from Streptomyces gulbargensis. Braz J Microbiol 2010;41:173-8.
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;37:387-93.
Narayana KJP, Kumar KG, Vijayalakshmi M. L-asparaginase production by Streptomyces albidoflavus. Indian J Microbiol 2008;48:331-6.
Richi VM, Vinod K, Vinoth R, Saurabh S, Prahlad CG, Rajendra KS. 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;9:e99037. Doi:10.1371/journal.pone.0099037. [Article in Press]
Dharmaraj S. Study of L-asparaginase production by Streptomyces noursei MTCC 10469, isolated from marine sponge Callyspongia diffusa. Iran J Biotechnol 2011;9:102-8.
Kavitha A, Vijayalakshmi M. Optimization and purification of L-asparaginase produced by Streptomyces tendae TK-VL_333. Z Naturforsch C: Biochem Biophys Biol Virol 2010;65:528-31.
El-Sabbagh SM, El-Batanony NH, Salem TA. L-asparaginase produced by Streptomyces strain isolated from egyptian soil: purification, characterization and its evaluation of the anti-tumor activity. Afr J Microbiol Res 2013;7:5677-86.