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Research article

Food Microbiology and Biotechnology

Biochemical Characterization of L-Asparaginase in NaCl-Tolerant Staphylococcus sp. OJ82 Isolated from Fermented Seafood

Sangwon Han 1, Jaejoon Jung 1 and Woojun Park 1*

Laboratory of Molecular Environmental Microbiology, Department of Environmental Science and Ecological Engineering, Korea University, Seoul 136-713, Republic of Korea

Received: May 9, 2014; Accepted: May 26, 2014

J. Microbiol. Biotechnol. 2014; 24(8): 1096-1104

Published August 28, 2014 https://doi.org/10.4014/jmb.1405.05021

Copyright © The Korean Society for Microbiology and Biotechnology.

Abstract

L-Asparaginase from gram-positive bacteria has been poorly explored. We conducted recombinant overexpression and purification of 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°C and over 90% of activity was retained between 37°C and 50°C, and its thermal stability range was narrower than that of commercial E. coli 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 Km 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

L-asparaginase, Purification, Staphylococcus, Jeotgal, Salt stress, Metal

References

  1. Boyd JW, Phillips AW. 1971. Purification and properties of L-asparaginase from Serratia marcescens. J. Bacteriol. 106: 578587.
  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.
    CrossRef
  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.
    Pubmed CrossRef
  4. Eisenberg H, Mevarech M, Zaccai G. 1992. Biochemical, structural and molecular genetic aspects of halophilism. Advan. Protein. Chem. 43: 1-61.
    CrossRef
  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.
    Pubmed CrossRef
  6. Felsenstein J. 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution. 39: 783-791.
    CrossRef
  7. Hendriksen HV, Kornbrust BA, Østergaard 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.
    Pubmed CrossRef
  8. Fisher SH, Wray LV Jr. 2002. Bacillus subtilis 168 contains two differentially regulated genes encoding L-asparaginase. J. Bacteriol. 184: 2148-2154.
    Pubmed CrossRef
  9. Gekil H, Gencer S. 2004. Production of L-Asparaginase in Enterobacter aerogenes expressing Vitreoscilla hemoglobin for efficient oxygen uptake. Appl. Microbiol. Biotechnol. 63: 691697.
  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.
    Pubmed CrossRef
  11. Kotzia GA, Labrou NE. 2005. Cloning, expression and characterisation of Erwinia carotovora L-asparaginase. J. Biotechnol. 119: 309-323.
    Pubmed CrossRef
  12. Kotzia GA, Lappa K, Labrou NE. 2007. Tailoring structurefunction properties of L-asparaginase: engineering resistance to trypsin cleavage. Biochem. J. 404: 337-343.
    Pubmed CrossRef
  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.
    CrossRef
  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.
  16. Masetti R, Pession A. 2009. First-line treatment of acute lymphoblastic leukemia with pegasparaginase. Biologics. 2:359-368.
    CrossRef
  17. Mesas JM, Gil JA, Martin JF.1990. Characterization and partial purification of L-asparaginase from Corynebacterium glutamicum. J. Gen. Microbiol. 136: 515-519.
    CrossRef
  18. Mevarech M, Frolow F, Gloss LM. 2000. Halophilic enzymes: proteins with a grain of salt. Biophys. Chem. 86:155-164.
    CrossRef
  19. Michalska K, Jakolski M. 2006. Structural aspects of Lasparaginases, their friends and relations. Acta. Biochim. Pol. 53: 627-640.
    Pubmed
  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.
    CrossRef
  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: 165173.
  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.
    Pubmed
  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.
  26. Stern ML, Phillips AW, Gottlieb AJ. 1976. Physical properties of L-asparaginase from Serratia marcescens. J. Bacteriol. 125:719-727.
    Pubmed
  27. Sung JS, Chun J, Choi S, Park W. 2012. Genome sequence of the halotolerant Staphylococcus sp. s train OJ82, i solated from Korean traditional salt fermented seafood. J. Bacteriol. 194:6353-6354.
    Pubmed CrossRef
  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.
    Pubmed CrossRef
  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: 27312739.
    Pubmed CrossRef
  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.
    Pubmed CrossRef
  32. Van den Berg H. 2011. Asparaginase revisited. Leuk. Lymphoma. 52: 168-178.
    Pubmed CrossRef
  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.
    Pubmed CrossRef
  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.
    Pubmed

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Article

Research article

J. Microbiol. Biotechnol. 2014; 24(8): 1096-1104

Published online August 28, 2014 https://doi.org/10.4014/jmb.1405.05021

Copyright © The Korean Society for Microbiology and Biotechnology.

Biochemical Characterization of L-Asparaginase in NaCl-Tolerant Staphylococcus sp. OJ82 Isolated from Fermented Seafood

Sangwon Han 1, Jaejoon Jung 1 and Woojun Park 1*

Laboratory of Molecular Environmental Microbiology, Department of Environmental Science and Ecological Engineering, Korea University, Seoul 136-713, Republic of Korea

Received: May 9, 2014; Accepted: May 26, 2014

Abstract

L-Asparaginase from gram-positive bacteria has been poorly explored. We conducted
recombinant overexpression and purification of 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°C and over 90% of activity was retained between 37°C and 50°C, and its thermal
stability range was narrower than that of commercial E. coli 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 Km 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: L-asparaginase, Purification, Staphylococcus, Jeotgal, Salt stress, Metal

References

  1. Boyd JW, Phillips AW. 1971. Purification and properties of L-asparaginase from Serratia marcescens. J. Bacteriol. 106: 578587.
  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.
    CrossRef
  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.
    Pubmed CrossRef
  4. Eisenberg H, Mevarech M, Zaccai G. 1992. Biochemical, structural and molecular genetic aspects of halophilism. Advan. Protein. Chem. 43: 1-61.
    CrossRef
  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.
    Pubmed CrossRef
  6. Felsenstein J. 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution. 39: 783-791.
    CrossRef
  7. Hendriksen HV, Kornbrust BA, Østergaard 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.
    Pubmed CrossRef
  8. Fisher SH, Wray LV Jr. 2002. Bacillus subtilis 168 contains two differentially regulated genes encoding L-asparaginase. J. Bacteriol. 184: 2148-2154.
    Pubmed CrossRef
  9. Gekil H, Gencer S. 2004. Production of L-Asparaginase in Enterobacter aerogenes expressing Vitreoscilla hemoglobin for efficient oxygen uptake. Appl. Microbiol. Biotechnol. 63: 691697.
  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.
    Pubmed CrossRef
  11. Kotzia GA, Labrou NE. 2005. Cloning, expression and characterisation of Erwinia carotovora L-asparaginase. J. Biotechnol. 119: 309-323.
    Pubmed CrossRef
  12. Kotzia GA, Lappa K, Labrou NE. 2007. Tailoring structurefunction properties of L-asparaginase: engineering resistance to trypsin cleavage. Biochem. J. 404: 337-343.
    Pubmed CrossRef
  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.
    CrossRef
  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.
  16. Masetti R, Pession A. 2009. First-line treatment of acute lymphoblastic leukemia with pegasparaginase. Biologics. 2:359-368.
    CrossRef
  17. Mesas JM, Gil JA, Martin JF.1990. Characterization and partial purification of L-asparaginase from Corynebacterium glutamicum. J. Gen. Microbiol. 136: 515-519.
    CrossRef
  18. Mevarech M, Frolow F, Gloss LM. 2000. Halophilic enzymes: proteins with a grain of salt. Biophys. Chem. 86:155-164.
    CrossRef
  19. Michalska K, Jakolski M. 2006. Structural aspects of Lasparaginases, their friends and relations. Acta. Biochim. Pol. 53: 627-640.
    Pubmed
  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.
    CrossRef
  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: 165173.
  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.
    Pubmed
  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.
  26. Stern ML, Phillips AW, Gottlieb AJ. 1976. Physical properties of L-asparaginase from Serratia marcescens. J. Bacteriol. 125:719-727.
    Pubmed
  27. Sung JS, Chun J, Choi S, Park W. 2012. Genome sequence of the halotolerant Staphylococcus sp. s train OJ82, i solated from Korean traditional salt fermented seafood. J. Bacteriol. 194:6353-6354.
    Pubmed CrossRef
  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.
    Pubmed CrossRef
  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: 27312739.
    Pubmed CrossRef
  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.
    Pubmed CrossRef
  32. Van den Berg H. 2011. Asparaginase revisited. Leuk. Lymphoma. 52: 168-178.
    Pubmed CrossRef
  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.
    Pubmed CrossRef
  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.
    Pubmed