Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
권호 표지
DOI QR코드

DOI QR Code

Biochemical Characterization of $\small{L}$-Asparaginase in NaCl-Tolerant Staphylococcus sp. OJ82 Isolated from Fermented Seafood

  • Han, Sangwon (Laboratory of Molecular Environmental Microbiology, Department of Environmental Science and Ecological Engineering, Korea University) ;
  • Jung, Jaejoon (Laboratory of Molecular Environmental Microbiology, Department of Environmental Science and Ecological Engineering, Korea University) ;
  • Park, Woojun (Laboratory of Molecular Environmental Microbiology, Department of Environmental Science and Ecological Engineering, Korea University)
  • Received : 2014.05.09
  • Accepted : 2014.05.26
  • Published : 2014.08.28
Download PDF
⟨ Previous Next ⟩

Abstract

$\small{L}$-Asparaginase from gram-positive bacteria has been poorly explored. We conducted recombinant overexpression and purification of $\small{L}$-asparaginase from Staphylococcus sp. OJ82 (SoAsn) isolated from Korean fermented seafood to evaluate its biotechnological potential as an antileukemic agent. SoAsn was expressed in Escherichia coli BL21 (DE3) with an estimated molecular mass of 37.5 kDa, determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Consistent with asparaginases in gram-negative bacteria, size-exclusion chromatography determined SoAsn as a homodimer. Interestingly, the optimal temperature of SoAsn was $37^{\circ}C$ and over 90% of activity was retained between $37^{\circ}C$ and $50^{\circ}C$, and its thermal stability range was narrower than that of commercial E. coli $\small{L}$-asparaginase (EcAsn). Both SoAsn and EcAsn were active between pH 9 and 10, although their overall pH-dependent enzyme activities were slightly different. The $K_m$ value of SoAsn was 2.2 mM, which is higher than that of EcAsn. Among eight metals tested for enzyme activity, cobalt and magnesium greatly enhanced the SoAsn and EcAsn activity, respectively. Interestingly, SoAsn retained more than 60% of its activity under 2 M NaCl condition, but the activity of EcAsn was reduced to 48%. Overall, the biochemical characteristics of SoAsn were similar to those of EcAsn, but its kinetics, cofactor requirements, and NaCl tolerance differed from those of EcAsn.

Keywords

References

  1. Boyd JW, Phillips AW. 1971. Purification and properties of L-asparaginase from Serratia marcescens. J. Bacteriol. 106: 578-587.
  2. Bradford MM. 1972. Rapid and sensitive method for quantitation of microgram quantities of protein utilizing principle of protein-dye binding. Anal. Biochem. 72: 248-254.
  3. Chohan SM, Rashid N. 2013. TK1656, a thermostable Lasparaginase from Thermococcus kodakaraensis, exhibiting highest ever reported enzyme activity. J. Biosci. Bioeng. 116: 438-443. https://doi.org/10.1016/j.jbiosc.2013.04.005
  4. Eisenberg H, Mevarech M, Zaccai G. 1992. Biochemical, structural and molecular genetic aspects of halophilism. Advan. Protein. Chem. 43: 1-61. https://doi.org/10.1016/S0065-3233(08)60553-7
  5. El-Bessoumy AA, Sarhan M, Mansour J. 2004. Production, isolation, and purification of L-asparaginase from Pseudomonas aeruginosa 50071 using solid-state fermentation. J. Biochem. Mol. Biol. 37: 387-393. https://doi.org/10.5483/BMBRep.2004.37.4.387
  6. Felsenstein J. 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution. 39: 783-791. https://doi.org/10.2307/2408678
  7. Hendriksen HV, Kornbrust BA, Ostergaard PR, Stringer MA. 2009. Evaluating the potential for enzymatic acrylamide mitigation in a range of food products using an asparaginase from Aspergillus oryzae. J. Agric. Food. Chem. 57: 4168-4176. https://doi.org/10.1021/jf900174q
  8. Fisher SH, Wray LV Jr. 2002. Bacillus subtilis 168 contains two differentially regulated genes encoding L-asparaginase. J. Bacteriol. 184: 2148-2154. https://doi.org/10.1128/JB.184.8.2148-2154.2002
  9. Gekil H, Gencer S. 2004. Production of L-Asparaginase in Enterobacter aerogenes expressing Vitreoscilla hemoglobin for efficient oxygen uptake. Appl. Microbiol. Biotechnol. 63: 691-697. https://doi.org/10.1007/s00253-003-1482-5
  10. Huang L, Liu Y, Sun Y, Yan Q, Jiang Z. 2014. Biochemical characterization of a novel L-asparaginase with low glutaminase activity from Rhizomucor miehei and its application in food safety and leukemia treatment. Appl. Environ. Microbiol. 80: 1591-1599.
  11. Kotzia GA, Labrou NE. 2005. Cloning, expression and characterisation of Erwinia carotovora L-asparaginase. J. Biotechnol. 119: 309-323. https://doi.org/10.1016/j.jbiotec.2005.04.016
  12. Kotzia GA, Lappa K, Labrou NE. 2007. Tailoring structurefunction properties of L-asparaginase: engineering resistance to trypsin cleavage. Biochem. J. 404: 337-343. https://doi.org/10.1042/BJ20061708
  13. Kumar DS, Sobba K. 2013. L-asparaginase from microbes: a comprehensive review. Adv. Biores. 3: 137-157.
  14. Li LZ, Xie TH, Li HJ, Qing C, Zhang GM, Suna MS. 2007. Enhancing the thermostability of E. coli L-asparaginase II by substitution with pro in predicted hydrogen-bonded turn structures. Enzyme. Microb. Technol. 41: 523-527. https://doi.org/10.1016/j.enzmictec.2007.04.004
  15. Magdy MY, Mohammad AA. 2008. Cloning, purification, characterization and immobilization of L-asparaginase II from E. coli W3110. Asian. J. Biochem. 3: 337-350. https://doi.org/10.3923/ajb.2008.337.350
  16. Masetti R, Pession A. 2009. First-line treatment of acute lymphoblastic leukemia with pegasparaginase. Biologics. 2: 359-368.
  17. Mesas JM, Gil JA, Martin JF.1990. Characterization and partial purification of L-asparaginase from Corynebacterium glutamicum. J. Gen. Microbiol. 136: 515-519. https://doi.org/10.1099/00221287-136-3-515
  18. Mevarech M, Frolow F, Gloss LM. 2000. Halophilic enzymes: proteins with a grain of salt. Biophys. Chem. 86: 155-164. https://doi.org/10.1016/S0301-4622(00)00126-5
  19. Michalska K, Jakolski M. 2006. Structural aspects of Lasparaginases, their friends and relations. Acta. Biochim. Pol. 53: 627-640.
  20. Mohapatra BR, Sani RK, Banerjee UC. 1995. Characterization of L-Asparaginase from Bacillus sp. isolated from an intertidal marine alga (Sargassum sp.). Lett. Appl. Microbiol. 21: 380-383. https://doi.org/10.1111/j.1472-765X.1995.tb01086.x
  21. Mohapatra BR, Sani RK, Banerjee UC. 1997. Production and properties of L-asparaginase from Mucor species associated with a marine sponge (Spirastrella sp.). Cytobios. 370: 165-173.
  22. Nagarethinam S, Nagappa, AN, Udupa N, Rao JV, Vanathi B MV. 2012. Microbial L-asparaginase and its future prospects. Asian. J. Med. Res. 1: 159-168.
  23. Nei M, Kumar S. 2000. Molecular evolution and phylogenetics. New York: Oxford University Press.
  24. Saitou N, Nei M. 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evolution. 4: 406-425.
  25. Pradhan B, Dash SK, Sahoo S. 2013. Screening and characterization of extracellular L-asparaginase producing Bacillus subtilis strain hswx88, isolated from Taptapani hotspring of Odishi, India. Asian. Pacific. J. Tropical. Biomed. 3: 936-941. https://doi.org/10.1016/S2221-1691(13)60182-3
  26. Stern ML, Phillips AW, Gottlieb AJ. 1976. Physical properties of L-asparaginase from Serratia marcescens. J. Bacteriol. 125: 719-727.
  27. Sung JS, Chun J, Choi S, Park W. 2012. Genome sequence of the halotolerant Staphylococcus sp. strain OJ82, isolated from Korean traditional salt fermented seafood. J. Bacteriol. 194: 6353-6354. https://doi.org/10.1128/JB.01653-12
  28. Suresh JV, Raju KJ. 2013. Studies on the production of Lasparaginase by aspergillus terreus MTCC 1782 using agroresidues under mixed substrate solid state fermentation. J. Chem. Bio. Phy. Sci. 3: 314-325.
  29. Swain AL, Jakolski M, Housset D, Rao JK, Woldawer A. 1993. Crystal structure of Escherichia coli L-asparaginase, an enzyme used in cancer therapy. Proc. Natl. Acad. Sci. 90: 1474-1478. https://doi.org/10.1073/pnas.90.4.1474
  30. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. 2011. MEGA5: Molecular Evolutionary Genetics Analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol. 28: 2731-2739. https://doi.org/10.1093/molbev/msr121
  31. Triantafillou DJ, Georgatsos JG, Kyriakidis DA. 1988. Purification and properties of a membrane-bound Lasparaginase of Tetrahymena pyriformis. Mol. Cell. Biochem. 81: 43-51.
  32. Van den Berg H. 2011. Asparaginase revisited. Leuk. Lymphoma. 52: 168-178. https://doi.org/10.3109/10428194.2010.537796
  33. Vidya J, Vasudevan M, Pandey A. 2014. Effect of surface charge alteration on stability of L-asparaginase II from E. coli sp. Enzyme. Microb. Technol. 56: 15-19. https://doi.org/10.1016/j.enzmictec.2013.12.012
  34. Yoshimoto T, Nishimura H, Saito Y, Sakurai K, Kamisaki Y, Wada H, et al. 1986. Characterization of polyethylene glycoL-modified L-asparaginase from Escherichia coli and its application to therapy for leukemia. Jpn. J. Cancer. Res. 77: 1264-1270.

Cited by

  1. Seeking efficacy in L-asparaginase to combat acute lymphoblastic leukemia (ALL): A review vol.9, pp.32, 2014, https://doi.org/10.5897/ajpp2015.4348
  2. A Newly Identified Glutaminase-Free L-Asparaginase (L-ASPG86) from the Marine Bacterium Mesoflavibacter zeaxanthinifaciens vol.26, pp.6, 2016, https://doi.org/10.4014/jmb.1510.10092
  3. Expression and purification of L-asparaginase from Escherichia coli and the inhibitory effects of cyclic dipeptides vol.31, pp.18, 2017, https://doi.org/10.1080/14786419.2016.1277350
  4. Insights into the Microbial L-Asparaginases: from Production to Practical Applications vol.20, pp.5, 2014, https://doi.org/10.2174/1389203720666181114111035
  5. A Comparative Study on Pseudomonal and Bacillus L. asparaginases. vol.13, pp.3, 2014, https://doi.org/10.22207/jpam.13.3.37
  6. A Comparative Study on Pseudomonal and Bacillus L. asparaginases. vol.13, pp.3, 2014, https://doi.org/10.22207/jpam.13.3.37
  7. Microbial l-asparaginase: purification, characterization and applications vol.202, pp.5, 2014, https://doi.org/10.1007/s00203-020-01814-1
  8. L-Asparaginases of Extremophilic Microorganisms in Biomedicine vol.14, pp.4, 2014, https://doi.org/10.1134/s1990750820040046
  9. Production, characterization and bioinformatics analysis of L -asparaginase from a new Stenotrophomonas maltophilia EMCC2297 soil isolate vol.10, pp.1, 2020, https://doi.org/10.1186/s13568-020-01005-7
  10. Biochemical characterization and thermodynamic principles of purified l-Asparaginase from novel Brevibacillus borstelensis ML12 vol.39, pp.None, 2014, https://doi.org/10.1016/j.bcab.2021.102260