Applied Microbiology and Biotechnology

, Volume 63, Issue 6, pp 691–697 | Cite as

Production of l-asparaginase in Enterobacter aerogenes expressing Vitreoscilla hemoglobin for efficient oxygen uptake

Original Paper

Abstract

This study is the first utilizing Vitreoscilla hemoglobin in a heterologous bacterium, Enterobacter aerogenes, to determine the effect of such a highly efficient oxygen-uptake system on the production of l-asparaginase, an enzyme that has attracted considerable attention due to its anti-tumor activity. Here, we show that the Vitreoscilla hemoglobin expressing strain has from 10-fold to more than two orders of magnitude lower l-asparaginase activity than the wild type or the control without the Vitreoscilla hemoglobin gene under different aeration conditions. Aeration and agitation were also determining factors for enzyme production. The enzyme activity was reduced considerably under both full aerobic and anaerobic conditions, while the highest enzyme activity was determined in cultures under low aeration and low agitation. Also, the effect of different concentrations of glucose on enzyme production showed catabolic repression. Glucose at 1% caused almost total inhibition of enzyme activity, while at 0.1% it showed a slightly stimulatory effect on enzyme production, compared with glucose-free medium.

Notes

Acknowledgement

This study was supported by a grant [TBAG2267(102T197)] from The Scientific and Technical Research Council of Turkey (TUBITAK).

References

  1. Abdel-Fattah YR, Olama ZA (2002) L-asparaginase production by Pseudomonas aeruginosa in solid-state culture: evaluation and optimization of culture conditions using factorial designs. Process Biochem 38:115–122Google Scholar
  2. Allison JP, Mandy WJ, Kitto GB (1971) The substrate specificity of l-asparaginase from Alcaligenes eutrophus. FEBS Lett 14:107–108PubMedGoogle Scholar
  3. Aung H-P, Bocola M, Schleper S, Röhm K-H (2000) Dynamics of a mobile loop at the active site of Escherichia coli asparaginase. Biochem Biophys Acta 1481:349–359PubMedGoogle Scholar
  4. Barnes WR, Dorn GL, Vela GR (1977) Effect of culture conditions on synthesis of l-asparaginase by Escherichia coli A-1. Appl Environ Microbiol 33:257–261PubMedGoogle Scholar
  5. Borek D, Jaskólski M (2001) Sequence analysis of enzymes with asparaginase activity. Acta Biochim Pol 48:893–902PubMedGoogle Scholar
  6. Carlsen M, Nielsen J (2001) Influence of carbon source on α-amylase production by Aspergillus aryzae. Appl Microbiol Biotechnol 57:346–349PubMedGoogle Scholar
  7. Cohen SN, Chang ACY, Hsu L (1972) Non-chromosomal antibiotic resistance in bacteria: genetic transformation of Escherichia coli by R-factor DNA. Proc Nat Acad Sci USA 69:2110–2114PubMedGoogle Scholar
  8. Dikshit KL, Webster DA (1988) Cloning, characterization, and expression of the bacterial globin gene from Vitreoscilla in Escherichia coli. Gene 70:377–386PubMedGoogle Scholar
  9. Dikshit RP, Dikshit KL, Liu YX, Webster DA (1992) The bacterial hemoglobin from Vitreoscilla can support the aerobic growth of Escherichia coli lacking terminal oxidases. Arch Biochem Biophys 293:241–245PubMedGoogle Scholar
  10. Ettinger LJ, Ettinger AG, Avramis VI, Gaynon PS (1997) Acute lymphoblastic leukaemia: a guide to asparaginase and pegaspargase therapy. BioDrugs 7:30–39Google Scholar
  11. Fisher SH, Wray Jr. LV (2002) Bacillus subtilis 168 contains two differentially regulated genes encoding l-asparaginase. J Bacteriol 184:2148–2154PubMedGoogle Scholar
  12. Geckil H, Stark BC, Webster DA (2001) Cell growth and oxygen uptake of Escherichia coli and Pseudomonas aeruginosa are differently effected by the genetically engineered Vitreoscilla hemoglobin gene. J Biotechnol 85:57–66PubMedGoogle Scholar
  13. Geckil H, Gencer S, Kahraman H, Erenler SO (2003) Genetic engineering of Enterobacter aerogenes with Vitreoscilla hemoglobin gene: cell growth, survival, and antioxidant enzyme status under oxidative stress. Res Microbiol 154:425–431PubMedGoogle Scholar
  14. Heinemann B, Howard AJ (1969) Production of tumor-inhibitory l-asparaginase by submerged growth of Serratia marscences. Appl Microbiol 18:550–554PubMedGoogle Scholar
  15. Hüser A, Klöppner U, Röhm K-H (1999) Cloning, sequence analysis, and expression of ansB from Pseudomonas fluorescens, encoding periplasmic glutaminase/asparaginase. FEMS Microbiol Lett 178:327–335CrossRefPubMedGoogle Scholar
  16. Jennings MP, Beacham IR (1993) Co-dependent positive regulation of the ansB promoter of Escherichia coli by CRP and the FNR protein: a molecular analysis. Mol Microbiol 9:155–164PubMedGoogle Scholar
  17. Joshi M, Dikshit KL (1994) Oxygen dependent regulation of Vitreoscilla globin gene: evidence for positive regulation by FNR. Biochem Biophys Res Commun 202:535–542PubMedGoogle Scholar
  18. Kelo E, Noronkoski T, Stoineva IB, Petkov DD, Mononen I (20002) β-Aspartylpeptides as substrates of l-asparaginases from Escherichia coli and Erwinia chrysanthemi. FEBS Lett 528:130–132CrossRefGoogle Scholar
  19. Khan AA, Pal SP, Raghavan SRV, Bhattacharyya PK (1970) Studies on Serratia marscences l-asparaginase. Biochem Biophys Res Comm 41:525–533PubMedGoogle Scholar
  20. Khosla C, Bailey JE (1989) Characterization of the oxygen-dependent promoter of the Vitreoscilla hemoglobin gene in Escherichia coli. J Bacteriol 171:5995–6004Google Scholar
  21. Kozak M, Jurga J (2002) A comparison between the crystal and solution structures of Escherichia coli asparaginase II. Acta Biochim Pol 49:509–513PubMedGoogle Scholar
  22. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193: 265–275Google Scholar
  23. Maladkar NK, Singh VK, Naik SR (1993) Fermentative production and isolation of l-asparaginase from Erwinia carotovora. Hind Antibiot Bull 35:77–86Google Scholar
  24. Messing J (1983) New M13 vectors for cloning. Methods Enzymol 101:20–78PubMedGoogle Scholar
  25. Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.Google Scholar
  26. Mukherjee J, Majumdar S, Scheper T (2000) Studies on nutritional and oxygen requirements for production of l-asparaginase by Enterobacter aerogenes. Appl Microbiol Biotechnol 53:180–184PubMedGoogle Scholar
  27. Müller HJ, Boos J (1998) Use of l-asparaginase in childhood ALL. Crit Rev Oncol Hematol 28:97–113PubMedGoogle Scholar
  28. Nawaz MS, Zhang D, Khan AA, Cerniglia CE (1998) Isolation and characterization of Enterobacter cloacae capable of metabolizing asparagine. Appl Microbiol Biotechnol 50:568–572PubMedGoogle Scholar
  29. Ortuño-Olea L, Durán-Vargas S (2000) The l-asparagine operon of Rhizobium etli contains a gene encoding an atypical asparaginase. FEMS Microbiol Lett 189:177–182PubMedGoogle Scholar
  30. Resnick AD, Magasanik B (1976) l-Asparaginase of Klebsiella aerogenes: activation of its synthesis by glutamine synthetase. J Biol Chem 251:2722–2728PubMedGoogle Scholar
  31. Roberts J (1976) Purification and properties of a highly potent antitumor glutaminase-asparaginase from Pseudomonas 7A. J Biol Chem 251:2119–2123PubMedGoogle Scholar
  32. Sun D, Setlow P (1991) Cloning, nucleotide sequence and expression of the Bacillus subtilis ans operon which codes for l-asparaginase and l-aspartase. J Bacteriol 173:3831–3845PubMedGoogle Scholar
  33. Tosa T, Sano R, Yamamoto K, Nakamura M, Ando K, Chibata I (1971) l-Asparaginase from Proteus vulgaris. Appl Microbiol 22:387–392PubMedGoogle Scholar
  34. Wei D Z, Liu H (1998) Promotion of l-asparaginase production by using n-dodecane. Biotechnol Tech 12:129–131Google Scholar
  35. Wriston JC Jr (1970) Asparaginase. Methods Enzymol 17:732–742Google Scholar

Copyright information

© Springer-Verlag 2004

Authors and Affiliations

  1. 1.Department of BiologyInonu UniversityMalatyaTurkey

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