Skip to main content
SpringerLink
Log in

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

  • Original Paper
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  • 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 38:115–122

    CAS  Google Scholar 

  • Allison JP, Mandy WJ, Kitto GB (1971) The substrate specificity of l-asparaginase from Alcaligenes eutrophus. FEBS Lett 14:107–108

    CAS  PubMed  Google Scholar 

  • 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 1481:349–359

    CAS  PubMed  Google Scholar 

  • Barnes WR, Dorn GL, Vela GR (1977) Effect of culture conditions on synthesis of l-asparaginase by Escherichia coli A-1. Appl Environ Microbiol 33:257–261

    CAS  PubMed  Google Scholar 

  • Borek D, Jaskólski M (2001) Sequence analysis of enzymes with asparaginase activity. Acta Biochim Pol 48:893–902

    CAS  PubMed  Google Scholar 

  • Carlsen M, Nielsen J (2001) Influence of carbon source on α-amylase production by Aspergillus aryzae. Appl Microbiol Biotechnol 57:346–349

    CAS  PubMed  Google Scholar 

  • 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 69:2110–2114

    CAS  PubMed  Google Scholar 

  • Dikshit KL, Webster DA (1988) Cloning, characterization, and expression of the bacterial globin gene from Vitreoscilla in Escherichia coli. Gene 70:377–386

    CAS  PubMed  Google Scholar 

  • 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 293:241–245

    CAS  PubMed  Google Scholar 

  • Ettinger LJ, Ettinger AG, Avramis VI, Gaynon PS (1997) Acute lymphoblastic leukaemia: a guide to asparaginase and pegaspargase therapy. BioDrugs 7:30–39

    CAS  Google Scholar 

  • Fisher SH, Wray Jr. LV (2002) Bacillus subtilis 168 contains two differentially regulated genes encoding l-asparaginase. J Bacteriol 184:2148–2154

    CAS  PubMed  Google Scholar 

  • 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 85:57–66

    CAS  PubMed  Google Scholar 

  • 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 154:425–431

    CAS  PubMed  Google Scholar 

  • Heinemann B, Howard AJ (1969) Production of tumor-inhibitory l-asparaginase by submerged growth of Serratia marscences. Appl Microbiol 18:550–554

    CAS  PubMed  Google Scholar 

  • 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 178:327–335

    Article  PubMed  Google Scholar 

  • 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 9:155–164

    CAS  PubMed  Google Scholar 

  • Joshi M, Dikshit KL (1994) Oxygen dependent regulation of Vitreoscilla globin gene: evidence for positive regulation by FNR. Biochem Biophys Res Commun 202:535–542

    CAS  PubMed  Google Scholar 

  • 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 528:130–132

    Article  Google Scholar 

  • Khan AA, Pal SP, Raghavan SRV, Bhattacharyya PK (1970) Studies on Serratia marscences l-asparaginase. Biochem Biophys Res Comm 41:525–533

    CAS  PubMed  Google Scholar 

  • Khosla C, Bailey JE (1989) Characterization of the oxygen-dependent promoter of the Vitreoscilla hemoglobin gene in Escherichia coli. J Bacteriol 171:5995–6004

    CAS  Google Scholar 

  • Kozak M, Jurga J (2002) A comparison between the crystal and solution structures of Escherichia coli asparaginase II. Acta Biochim Pol 49:509–513

    CAS  PubMed  Google Scholar 

  • Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193: 265–275

    CAS  Google Scholar 

  • Maladkar NK, Singh VK, Naik SR (1993) Fermentative production and isolation of l-asparaginase from Erwinia carotovora. Hind Antibiot Bull 35:77–86

    CAS  Google Scholar 

  • Messing J (1983) New M13 vectors for cloning. Methods Enzymol 101:20–78

    CAS  PubMed  Google Scholar 

  • Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.

  • Mukherjee J, Majumdar S, Scheper T (2000) Studies on nutritional and oxygen requirements for production of l-asparaginase by Enterobacter aerogenes. Appl Microbiol Biotechnol 53:180–184

    CAS  PubMed  Google Scholar 

  • Müller HJ, Boos J (1998) Use of l-asparaginase in childhood ALL. Crit Rev Oncol Hematol 28:97–113

    PubMed  Google Scholar 

  • Nawaz MS, Zhang D, Khan AA, Cerniglia CE (1998) Isolation and characterization of Enterobacter cloacae capable of metabolizing asparagine. Appl Microbiol Biotechnol 50:568–572

    CAS  PubMed  Google Scholar 

  • Ortuño-Olea L, Durán-Vargas S (2000) The l-asparagine operon of Rhizobium etli contains a gene encoding an atypical asparaginase. FEMS Microbiol Lett 189:177–182

    PubMed  Google Scholar 

  • Resnick AD, Magasanik B (1976) l-Asparaginase of Klebsiella aerogenes: activation of its synthesis by glutamine synthetase. J Biol Chem 251:2722–2728

    CAS  PubMed  Google Scholar 

  • Roberts J (1976) Purification and properties of a highly potent antitumor glutaminase-asparaginase from Pseudomonas 7A. J Biol Chem 251:2119–2123

    CAS  PubMed  Google Scholar 

  • 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 173:3831–3845

    CAS  PubMed  Google Scholar 

  • Tosa T, Sano R, Yamamoto K, Nakamura M, Ando K, Chibata I (1971) l-Asparaginase from Proteus vulgaris. Appl Microbiol 22:387–392

    CAS  PubMed  Google Scholar 

  • Wei D Z, Liu H (1998) Promotion of l-asparaginase production by using n-dodecane. Biotechnol Tech 12:129–131

    CAS  Google Scholar 

  • Wriston JC Jr (1970) Asparaginase. Methods Enzymol 17:732–742

    CAS  Google Scholar 

Download references

Acknowledgement

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

Author information

Authors and Affiliations

  1. Department of Biology, Inonu University, 44069, Malatya, Turkey

    H. Geckil & S. Gencer

Authors
  1. H. Geckil
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. S. Gencer
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to H. Geckil.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Geckil, H., Gencer, S. Production of l-asparaginase in Enterobacter aerogenes expressing Vitreoscilla hemoglobin for efficient oxygen uptake. Appl Microbiol Biotechnol 63, 691–697 (2004). https://doi.org/10.1007/s00253-003-1482-5

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-003-1482-5

Keywords

Search

Navigation