l-asparaginase is a widely used cancer chemotherapy enzyme. The source for the enzyme with this property is mainly bacterial and its synthesis is strongly regulated by oxygen. In this study, we utilized two recombinant systems: one carried the gene (vgb) for the Vitreoscilla hemoglobin (VHb), a protein of prokaryotic origin which confers a highly efficient oxygen uptake to its host and the other carried the l-asparaginase gene (ansB). The host bacteria were Escherichia coli, Enterobacter aerogenes, and Pseudomonas aeruginosa. Of these three bacteria, all gram-negative, E. coli and its recombinant strain showed up to sevenfold higher l-asparaginase activity in lactose than in other carbon sources. Although, in this bacterium glycerol was the poorest source for l-asparaginase synthesis, it supported the highest biomass production. In glucose medium, l-asparaginase activity of E. aerogenes was about threefold higher than its vgb and ansB recombinants. ansB recombinant showed significantly higher enzyme levels than both host and vgb recombinants in glycerol and lactose media. In this bacterium, VHb/vgb clearly caused a decrease in the enzyme synthesis under all conditions. As seen for E. aerogens, glycerol was the most favorable carbon source for P. aeruginosa and its vgb strain in terms of both l-asparaginase synthesis and biomass production. The cultures grown in glycerol had more than two- and threefold biomass than in glucose and lactose, respectively, and up to elevenfold than in mannitol. Indeed, the highest biomass production for all bacteria and their recombinants was in glycerol. The VHb/vgb system is clearly advantageous for production of l-asparaginase in P. aeruginosa. The same, however, does not hold true for E. aerogenes.
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Ettinger, L. J., Ettinger, A. G., Avramis, V. I., & Gaynon, P. S. (1997). Acute lymphoblastic leukaemia: a guide to asparaginase and pegaspargase therapy. Biodrugs, 7, 30–39.
Geckil, H., & Gencer, S. (2004). Production of l-asparaginase in Enterobacter aerogenes expressing Vitreoscilla hemoglobin for efficient oxygen uptake. Applied Microbiology and Biotechnology, 63, 691–697.
Mukherjee, J., Majumdar, S., & Scheper, T. (2000). Studies on nutritional and oxygen requirements for production of l-asparaginase by Enterobacter aerogenes. Applied Microbiology and Biotechnology, 53, 180–184.
Lee, S. M., Ross, J. T., Gustafson, M. E., Wroble, M. H., & Muschik, G. M. (1986). Large-scale recovery and purification of l-asparaginase from Erwinia carotovora. Applied Microbiology and Biotechnology, 12, 229–247.
Cornea, C. P., Lupescu, I., Vatafu, I., Savoiu, V. G., & Campeanu, G. H. (2000). Isolation and characterization of some L-asparaginase producing recombinant Escherichia coli strains. Roumamian Biotechnological Letters, 5, 471–478.
Heinemann, B., & Howard, A. J. (1969). Production of tumor-inhibitory l-asparaginase by submerged growth of Serratia marcescens. Applied Microbiology, 18, 64–67.
Muller, H. J., & Boos, J. (1998). Use of L-asparaginase in childhood ALL. Critical Review in Oncology Hematology, 28, 97–113.
Borek, D., & Jaskolski, M. (2001). Sequence analysis of enzymes with asparaginase activity. Acta Biochimica Polonica, 48, 893–902.
Kozak, M., & Jurga, S. (2002). A comparison between the crystal and solution structures of Escherichia coli asparaginase II. Acta Biochimica Polonica, 49, 509–513.
Ohnuma, T., Holland, J. F., & Freeman, A. (1970). Biochemical and pharmacological studies with asparaginase in man. Cancer Research, 30, 2297–305.
Swain, A. L., Jaskolski, M., Housset, D., Rao, J. K. M., & Wlodawer, A. (1993). Crystal structure of Escherichia coli l-asparaginase, an enzyme used in cancer therapy. Proceedings of theNational Academy of Sciences USA, 90, 1474–1478.
Fisher, S. H., & Wray, L. V. (2002). Bacillus subtilis 168 contains two differentially regulated genes encoding l-asparaginase. Journal of Bacteriology, 184, 2148–2154.
Jennings, M. P., & Beacham, I. R. (1990). Analysis of the Escherichia coli gene encoding l-asparaginase II, ansB, and its regulation by cyclic AMP receptor and FNR proteins. Journal of Bacteriology, 172, 1491–1498.
Barnes, W. R., Dorn, G. L., & Vela, G. R. (1977). Effect of culture conditions on synthesis of l-asparaginase by Escherichia coli A-1. Applied and Environmental Microbiology, 33, 257–261.
El-Bessoumy, A. A., Sarhan, M., & Mansour, J. (2004). Production, isolation, and purification of l-asparaginase from Pseudomonas aeruginosa 50071 using solid-state fermentation. Journal of Biochemistry and Molecular Biology, 37, 387–393.
Geckil, H., Gencer, S., Kahraman, H., & Erenler, S. O. (2003). Genetic engineering of Enterobacter aerogenes with the Vitreoscilla hemoglobin gene: cell growth, survival, and antioxidant enzyme status under oxidative stress. Research in Microbiology, 154, 425–431.
Geckil, H., Stark, B. C., & Webster, D. A. (2001). Cell growth and oxygen uptake of Escherichia coli and Pseudomonas aeruginosa are differently effected by the genetically engineered Vitreoscilla hemoglobin gene. Journal of Biotechnology, 85, 57–66.
Khosla, C., & Bailey, J. E. (1988). Heterologous expression of a bacterial haemoglobin improves the growth properties of recombinant Escherichia coli. Nature, 331, 633–635.
Stark, B. C., Webster, D. A., & Dikshit, K. L. (1999). Vitreoscilla hemoglobin: molecular biology, biochemistry, and practical applications. Recent Reseach Development in Biotechnology and Bioengineering, 2.
Park, K. W., Kim, K. J., Howard, A. J., Stark, B. C., & Webster, D. A. (2002). Vitreoscilla hemoglobin binds to subunit I of cytochrome bo ubiquinol oxidases. The Journal of Biological Chemistry, 277, 33334–33337.
Ramandeep, H. K. W., Raje, M., Kim, K. J., Stark, B. C., Dikshit, K. L., & Webster, D. A. (2001). Vitreoscilla hemoglobin. Intracellular localization and binding to membranes. The Journal of Biological Chemistry, 276, 24781–24789.
Joshi, M., & Dikshit, K. L. (1994). Oxygen-Dependent Regulation of Vitreoscilla globin gene: evidence for positive regulation by FNR. Biochemical Biophysical Reseach Communication, 202, 535–542.
Dikshit, R. P., Dikshit, K. L., Liu, Y. X., & Webster, D. A. (1992). The Bacterial hemoglobin from Vitreoscilla can support the aerobic growth of Escherichia coli lacking terminal oxidases. Archives of Biochemistry and Biophysics, 293, 241–245.
Dikshit, K. L., & Webster, D. A. (1988). Cloning, Characterization and expression of the bacterial globin gene from Vitreoscilla in Escherichia coli. Gene, 70, 377–386.
Erenler, S. O., Gencer, S., Geckil, H., Stark, B. C., & Webster, D. A. (2004). Cloning and expression of the Vitreoscilla hemoglobin gene in Enterobacter aerogenes: effect on cell growth and oxygen uptake. Appl Biochem Micro, 40, 241–248.
Ortuno-Olea, L., & Duran-Vargas, S. (2000). The l-asparagine operon of Rhizobium etli contains a gene encoding an atypical asparaginase. Fems Microbiology Letters, 189, 177–182.
Chung, J. W., Webster, D. A., Pagilla, K. R., & Stark, B. C. (2001). Chromosomal integration of the Vitreoscilla hemoglobin gene in Burkholderia and Pseudomonas for the purpose of producing stable engineered strains with enhanced bioremediating ability. Journal of Industrial Microbiology & Biotechnology, 27, 27–33.
Huser, A., Kloppner, U., & Rohm, K. H. (1999). Cloning, sequence analysis, and expression of ansB from Pseudomonas fluorescens, encoding periplasmic glutaminase/asparaginase. Fems Microbiology Letters, 178, 327–335.
Geckil, H., Ates, B., Gencer, S., Uckun, M., & Yilmaz, I. (2005). Membrane permeabilization of gram-negative bacteria with a potassium phosphate/hexane aqueous phase system for the release of l-asparaginase: an enzyme used in cancer therapy. Process Biochemistry, 40, 573–579.
Sambrook, J., & Russell, D. W. (2001). Molecular cloning : a laboratory manual (3rd ed.). Cold Spring Harbor: Cold Spring Harbor Laboratory Press.
Narta, U., Roy, S., Kanwar, S. S., & Azmi, W. (2011). Improved production of l-asparaginase by Bacillus brevis cultivated in the presence of oxygen-vectors. Bioresource Technology, 102, 2083–2085.
Agarwal, A., Kumar, S., & Veeranki, V. D. (2011). Effect of chemical and physical parameters on the production of l-asparaginase from a newly isolated Serratia marcescens SK-07. Letters in Applied Microbiology, 52, 307–313.
Boerman, S., Webster, D. A., & Roush, A. H. (1981). Control of heme content in Vitreoscilla by oxygen. Federation Proceedings, 40, 1864–1864.
Khleifat, K. M. (2006). Correlation between bacterial hemoglobin and carbon sources: their effect on copper uptake by transformed E. coli strain alpha DH5. Current Microbiology, 52, 64–68.
Geckil, H., Gencer, S., & Uckun, M. (2004). Vitreoscilla hemoglobin expressing Enterobacter aerogenes and Pseudomonas aeruginosa respond differently to carbon catabolite and oxygen repression for production of l-asparaginase, an enzyme used in cancer therapy. Enzyme and Microbial Technology, 35, 182–189.
Hymavathi, M., Sathish, T., Brahmaiah, P., & Prakasham, R. S. (2010). Impact of carbon and nitrogen sources on l-asparaginase production by isolated Bacillus circulans (MTCC 8574): application of saturated Plackett-Burman Design. Chemical and Biochemical Engineering Quarterly, 24, 473–480.
Wei, D. Z., & Liu, H. (1998). Promotion of L-asparaginase production by using n-dodecane. Biotechnology Techniques, 12, 129–131.
Wriston, J. C., Jr., & Yellin, T. O. (1973). L-asparajinase: a review. Advances in Enzymology and Related Areas of Molecular Biology, 39, 185–248.
Willis, R. C., & Woolpolk, C. A. (1974). Asparagine utilization in Escherichia coli. Journal Bacteriology, 118, 231–41.
Golden, K. J., & Bernlohr, R. W. (1985). Nitrogen catabolite repression of the l-asparaginase of Bacillus licheniformis. Journal of Bacteriology, 164, 938–940.
Treshalina, H. M., Lukasheva, E. V., Sedakova, L. A., Firsova, G. A., Guerassimova, G. K., Gogichaeva, N. V., & Berezov, T. T. (2000). Anticancer enzyme l-lysine α-oxidase. Applied Biochemistry Biotechnology, 88, 267–273.
Roth, G., Nunes, J. E. S., Rosado, L. A., Bizarro, C. V., Volpato, G., Nunes, C. P., Renard, G., Basso, L. A., Santos, D. S., & Chies, J. M. (2013). Recombinant Erwinia carotovora l-asparaginase II production in Escherichia coli fed-batch cultures. Brazilian Journal of Chemical Engineering, 30, 245–256.
Uppuluri, K. B., & Reddy, D. S. R. (2009). Optimization of l-asparaginase production by Isolated Aspergillus niger using sesame cake in a column bioreactor. Journal Pure Applied Microbiology, 3, 83–90.
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Erenler, S.O., Geckil, H. Effect of Vitreoscilla Hemoglobin and Culture Conditions on Production of Bacterial l-Asparaginase, an Oncolytic Enzyme. Appl Biochem Biotechnol 173, 2140–2151 (2014). https://doi.org/10.1007/s12010-014-1016-x
- Vitreoscilla hemoglobin
- Chemotherapeutic enzymes