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Biologia




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Volume 69, Issue 12

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Optimization and purification of anticancer enzyme L-glutaminase from Alcaligenes faecalis KLU102

Sureshbabu Pandian / Venkataraman Deepak
  • Department of Biotechnology, Kalasalingam University, Krishnankoil, 626126, Tamilnadu, India
  • School of Science and Technology, Nottingham Trent University, Nottingham, NG11 8NS, UK
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/ Shiva Sivasubramaniam
  • Department of Biotechnology, Kalasalingam University, Krishnankoil, 626126, Tamilnadu, India
  • School of Science and Technology, Nottingham Trent University, Nottingham, NG11 8NS, UK
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/ Hariharan Nellaiah / Krishnan Sundar
Published Online: 2015-01-06 | DOI: https://doi.org/10.2478/s11756-014-0486-1

Abstract

L-Glutaminase, an amidohydrolase, is gaining importance on account of its potential anticancer activity. L-Glutaminase produced by Alcaligenes faecalis KLU102, isolated from the marine realm (Bay of Bengal), exhibits potential anticancer activity. Response surface methodology was employed for optimizing the medium composition. The concentrations of the various constituents were as follows: arabinose (2%), skim milk (4%), and salts viz. K2HPO4, KH2PO4, MgSO4, and NaCl (2%). The bacterium grown in the optimized medium yielded an enzyme activity of 1.34 ± 0.07 IU/mg in shake-flask cultures and this doubled (2.77 ± 0.35 IU/mg) when scale-up studies were conducted using a 3-L fermenter. The enzyme was purified to homogeneity using ion-exchange chromatography, and the purified enzyme was found to have a specific activity of 54.72 IU/mg, with a molecular weight of 37 kDa. Immobilization of the enzyme on PEG-PHB nanoparticles improved the stability of the enzyme significantly. Purified L-glutaminase exhibited cytotoxic activity against HeLa cells, as assayed by the MTT assay, with an IC50 value of 12.5 μg/mL.

Keywords: L-glutaminase; response surface methodology; PEG-PHB nanoparticle; anticancer

  • [1] Anastasiou D. & Cantley L.C. 2012. Breathless cancer cells get fat on glutamine. Cell Res. 22: 443–446. http://dx.doi.org/10.1038/cr.2012.5CrossrefGoogle Scholar

  • [2] Arockiasamy S. & Banik R.M. 2008. Optimization of gellan gum production by Sphingomonas paucimobilis ATCC 31461 with non-ionic surfactants using central composite design. J. Biosci. Bioeng. 105: 204–210. http://dx.doi.org/10.1263/jbb.105.204Web of ScienceGoogle Scholar

  • [3] Banik R.M., Santhiagu A. & Upadhyay S.N. 2007. Optimization of nutrients for gellan gum production by Sphingomonas paucimobilis ATCC-31461 in molasses based medium using response surface methodology. Bioresour. Technol. 98: 792–797. http://dx.doi.org/10.1016/j.biortech.2006.03.012CrossrefWeb of ScienceGoogle Scholar

  • [4] Chan R.T., Marcal H., Ahmed T., Russell R.A., Holden P.J. & Foster L.J.R. 2013. Poly(ethylene glycol)-modulated cellular biocompatibility of polyhydroxyalkanoate films. Polym. Int. 62: 884–892. http://dx.doi.org/10.1002/pi.4451CrossrefWeb of ScienceGoogle Scholar

  • [5] Chandrasekaran M. 1997. Industrial enzymes from marine microorganisms — Indian scenario. J. Mar. Biotechnol. 5: 86–89. Google Scholar

  • [6] Chen J. & Herrup K. 2012. Glutamine acts as a neuroprotectant against DNA damage, β-amyloid and H2O2-induced stress. PLoS One 7: e33177. http://dx.doi.org/10.1371/journal.pone.0033177CrossrefGoogle Scholar

  • [7] Criss W.E. 1971. A review of isozymes in cancer. Cancer Res. 31: 1523–1542. Google Scholar

  • [8] Ghosh S., Chaganti S.R. & Prakasham R.S. 2012. Polyaniline nanofiber as a novel immobilization matrix for the anti-leukemia enzyme L-asparaginase. J. Mol. Catal. B Enzym. 74: 132–137. http://dx.doi.org/10.1016/j.molcatb.2011.09.009CrossrefWeb of ScienceGoogle Scholar

  • [9] Gulati R., Saxena R.K. & Gupta R. 1997. A rapid plate assay for screening L-asparaginase producing microorganisms. Lett. Appl. Microbiol. 24: 23–26. http://dx.doi.org/10.1046/j.1472-765X.1997.00331.xCrossrefGoogle Scholar

  • [10] Hans M.L. & Lowmanm A.M. 2002. Biodegradable nanoparticles for drug delivery and targeting. Curr. Opin. Solid State Mater. Sci. 6: 319–327. http://dx.doi.org/10.1016/S1359-0286(02)00117-1CrossrefGoogle Scholar

  • [11] Hartman S.C. 1971. Glutaminases and γ-glutamyltransferases, pp. 79–100. In: Boyer P.D. (ed.) The Enzymes, Academic Press, New York. Google Scholar

  • [12] Holt J.G., Krieg N.R., Sneath P.H.A., Staley J.T. & Williams S.T. 1994. Bergey’s Manual of Determinative Bacteriology. 9th Ed., Williamsons and Wilkins, Baltimore. Google Scholar

  • [13] Imada A., Igarasi S., Nakahama K. & Isono M. 1973. Asparaginase and glutaminase activities of microorganism. J. Gen. Microbiol. 76: 85–99. http://dx.doi.org/10.1099/00221287-76-1-85CrossrefGoogle Scholar

  • [14] Iyer P. & Singhal R.S. 2008. Production of glutaminase (EC 3.2.1.5) from Zygosaccharomyces rouxii: statistical optimization using response surface methodology. Bioresour. Technol. 99: 4300–4307. http://dx.doi.org/10.1016/j.biortech.2007.08.076CrossrefWeb of ScienceGoogle Scholar

  • [15] Jobmann M. & Rafler G. 2002. Submicronparticles from biodegradable polymers. Int. J. Pharm. 242: 213–217. http://dx.doi.org/10.1016/S0378-5173(02)00160-6CrossrefGoogle Scholar

  • [16] Knox W.E., Tremblay G.C., Spanier B.B. & Friedell G.H. 1967. Glutaminase activities in normal and neoplastic tissues of the rat. Cancer Res. 27: 1456–1458. Google Scholar

  • [17] Li X., Liu K.L., Li J., Tan E.P., Chan L.M., Lim C.T. & Goh S.H. 2006. Synthesis, characterization, and morphology studies of biodegradable amphiphilic poly[(R)-3-hydroxybutyrate]-alt-poly(ethylene glycol) multiblock copolymers. Biomacromolecules 7: 3112–3119. http://dx.doi.org/10.1021/bm060675fCrossrefGoogle Scholar

  • [18] Lu W., Pelicano H. & Huang P. 2010. Cancer metabolism: is glutamine sweeter than glucose?. Cancer Cell 18: 199–200. http://dx.doi.org/10.1016/j.ccr.2010.08.017CrossrefWeb of ScienceGoogle Scholar

  • [19] Nandakumar R., Yoshimune K., Wakayama M. & Moriguchi M. 2003. Microbial glutaminase: biochemistry, molecular approaches and applications in the food industry. J. Mol. Catal. B Enzym. 23: 87–100. http://dx.doi.org/10.1016/S1381-1177(03)00075-4CrossrefGoogle Scholar

  • [20] Padma I. & Singhal R. 2010. Isolation, screening, and selection of an L-glutaminase producer from soil and media optimization using a statistical approach. Biotechnol. Bioprocess. Eng. 15: 975–983. http://dx.doi.org/10.1007/s12257-009-0187-8CrossrefWeb of ScienceGoogle Scholar

  • [21] Pandian S.R., Deepak V., Kalishwaralal K., Rameshkumar N., Jeyaraj M. & Gurunathan S. 2010. Optimization and fed-batch production of PHB utilizing dairy waste and seawater as nutrient sources by Bacillus megaterium SRKP-3. Bioresour. Technol. 101: 705–711. http://dx.doi.org/10.1016/j.biortech.2009.08.040CrossrefGoogle Scholar

  • [22] Pandian S.R.K., Deepak V., Kalimuthu K., Muniyandi J., Rameshkumar N. & Gurunathan S. 2009. Synthesis of PHB nanoparticles from optimized medium utilizing dairy industrial waste using Brevibacterium casei SRKP2: a green chemistry approach. Colloids Surf. B Biointerfaces 74: 266–273. http://dx.doi.org/10.1016/j.colsurfb.2009.07.029Web of ScienceCrossrefGoogle Scholar

  • [23] Payne R.W., Murphy B.M. & Manning M.C. 2011. Product development issues for PEGylated proteins. Pharm. Dev. Technol. 16: 423–440. http://dx.doi.org/10.3109/10837450.2010.513990CrossrefWeb of ScienceGoogle Scholar

  • [24] Pedreschi F., Kaack K. & Granby K. 2008. The effect of asparaginase on acrylamide formation in French fries. Food Chem. 109: 386–392. http://dx.doi.org/10.1016/j.foodchem.2007.12.057CrossrefGoogle Scholar

  • [25] Reitzer L.J., Wice B.M. & Kennell D. 1979. Evidence that glutamine, not sugar, is the major energy source for cultured HeLa cells. J. Biol. Chem. 254: 2669–2676. Google Scholar

  • [26] Sarkar S., Pramanik A., Mitra A. & Mukherjee J. 2010. Bioprocessing data for the production of marine enzymes. Mar. Drugs 8: 1323–1372. http://dx.doi.org/10.3390/md8041323Web of ScienceCrossrefGoogle Scholar

  • [27] Singh P. & Banik R. M. 2013. Biochemical characterization and antitumor study of L-glutaminase from Bacillus cereus MTCC 1305. Appl. Biochem. Biotechnol. 171: 522–531. http://dx.doi.org/10.1007/s12010-013-0371-3CrossrefGoogle Scholar

  • [28] Soares A. L., Guimaraes G. M., Polakiewicz B., de Moraes Pitombo R. N. & Abrahao-Neto J. 2002. Effects of polyethylene glycol attachment on physicochemical and biological stability of E. coli L-asparaginase. Int. J. Pharm. 237: 163–170. http://dx.doi.org/10.1016/S0378-5173(02)00046-7CrossrefGoogle Scholar

  • [29] Souba W.W. 1993. Glutamine and cancer. Ann. Surg. 218: 715–728. http://dx.doi.org/10.1097/00000658-199312000-00004CrossrefGoogle Scholar

  • [30] Tamber H., Johansen P., Merkle H.P. & Gander B. 2005. Formulation aspects of biodegradable polymeric microspheres for antigen delivery. Adv. Drug Deliver. Rev. 57: 357–376. http://dx.doi.org/10.1016/j.addr.2004.09.002CrossrefGoogle Scholar

  • [31] Thompson J.D., Gibson T.J., Plewniak F., Jeanmougin F. & Higgins D.G. 1997. The CLUSTAL X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 25: 4876–4882. http://dx.doi.org/10.1093/nar/25.24.4876CrossrefGoogle Scholar

  • [32] Wade H.E. 1980. Synthesis and functions of microbial asparaginases and glutaminases, pp. 563–575. In: Payne J.W. (ed.), Microorganisms and Nitrogen Sources, John Wiley and Sons, New York. Google Scholar

  • [33] Wakayama M., Yamagata T., Kamemura A., Bootim N., Yano S., Tachiki T., Yoshimune K. & Moriguchi M. 2005. Characterization of salt-tolerant glutaminase from Stenotrophomonas maltophilia NYW-81 and its application in Japanese soy sauce fermentation. J. Ind. Microbiol. Biotechnol. 32: 383–390. http://dx.doi.org/10.1007/s10295-005-0257-7CrossrefGoogle Scholar

  • [34] Wise D.R. & Thompson C.B. 2010. Glutamine addiction: a new therapeutic target in cancer. Trends Biochem. Sci. 35: 427–433. http://dx.doi.org/10.1016/j.tibs.2010.05.003Web of ScienceCrossrefGoogle Scholar

  • [35] Zinn M., Witholt B. & Egli T. 2001. Occurrence, synthesis and medical application of bacterial polyhydroxyalkanoate. Adv. Drug Deliv. Rev. 53: 5–21. http://dx.doi.org/10.1016/S0169-409X(01)00218-6CrossrefGoogle Scholar

About the article

Published Online: 2015-01-06

Published in Print: 2014-12-01


Citation Information: Biologia, Volume 69, Issue 12, Pages 1644–1651, ISSN (Online) 1336-9563, ISSN (Print) 0006-3088, DOI: https://doi.org/10.2478/s11756-014-0486-1.

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© 2014 Slovak Academy of Sciences. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License. BY-NC-ND 3.0

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