Recombinant Formaldehyde Dehydrogenase and Gene-Engineered Methylotrophic Yeasts as Bioanalitycal Instruments for Assay of Toxic Formaldehyde

  • Galyna Gayda
  • Olha Demkiv
  • Mykhailo Gonchar
  • Solomiya Paryzhak
  • Wolfgang Schuhmann
Part of the NATO Science for Peace and Security Series A: Chemistry and Biology book series (NAPSA)


Recombinant yeast clones, originated from the recipient Hansenula polymorpha strains NCYC 495 and CBS 4732, resistent to elevated concentrations of formaldehyde in a medium (up to 15–20 mM) and overproducing a homologous NAD- and glutathione-dependent form-aldehyde dehydrogenase, were constructed. Optimal cultivation conditions for the highest yield of the enzyme were established. A simple scheme for the isolation of formaldehyde dehydrogenase from the re-combinant strains was proposed, and some characteristics of the purified enzyme were studied. Enzymatic and biosensoric methods for formaldehyde assay based on the formaldehyde dehydrogenase and the constructed recombinant cells were developed. The reliability of the developed analytical approaches was tested on real samples of waste waters, pharmaceuticals, formaldehyde-containing industrial products, and vaccines. The comparison of formaldehyde content values obtained by the use of biosensors (enzyme and cells-based), enzymatic methods and two routinely used chemical ones (chromotropic acid and 3-methyl-2-benzothiazolinone hydrazone) showed a good correlation between these approaches.


Yeast Hansenula polymorpha (Pichia angustagene engineering formaldehyde dehydrogenase enzymatic assay biosensor formaldehyde 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Allais, J.J., Louktibi, A., and Baratti, J., 1983, Oxidation of methanol by the yeast, Pichia pastoris, purification and properties of the formaldehyde dehydrohenase, Agric. Biol. Chem. 47 (7): 1509-1516.Google Scholar
  2. Baerends, R.J.S., Sulter, G.J., Jeffries, T.W., Cregg, J.M., and Veenhuis, M., 2002, Molecular characterization of the Hansenula polymorpha FLD1 gene encoding formaldehyde dehydrogenase, Yeast 19: 37-42.CrossRefPubMedGoogle Scholar
  3. Ben Ali, M., Korpan, Y., Gonchar, M., El’skaya, A., Maaref, M.A., Jaffrezic-Renault, N., and Martelet, C., 2006, Formaldehyde assay by capacitance versus voltage and impe-dance measurements using be-layer bio-recognition membrane. Biosens. Bioelectron. 22: 575-581.CrossRefPubMedGoogle Scholar
  4. Ben Ali, M., Gonchar, M., Gayda, G., Paryzhak, S., Maaref, M.A., Jaffrezic-Renault, N., and Korpan, Y., 2007, Formaldehyde-sensitive sensor based on recombinant formaldehyde dehydrogenase using capacitance versus voltage measurements, Biosens. Bioelectron. 22 (12): 2790-2795.CrossRefPubMedGoogle Scholar
  5. Delorme, E., 1989, Transformation of Saccharomyces cerevisiae by electroporation, Appl. Environ. Microbiol. 55 (9): 2242-2246.PubMedGoogle Scholar
  6. Demkiv, O.M., Paryzhak, S.Ya., Krasovs’ka, ȿ.S., Stasyk, Ɉ.V., Gayda, G.Z., Sibirny, A.A., and Gonchar, Ɇ.V., 2005, Construction of methylotrophic yeast Hansenula polymorpha strains overproducing formaldehyde dehydrogenase, Biopolymers and cell. 21(6): 525-530 (in Ukrainian).Google Scholar
  7. Demkiv, O., Paryzhak, S., Gayda, G., Sibirny V., and Gonchar M.V., 2007a, Formaldehyde dehydrogenase from the recombinant yeast Hansenula polymorpha: ɿsolation and bioanalytic application, FEMS Yeast Research. 7: 1153-1159.CrossRefPubMedGoogle Scholar
  8. Demkiv, O., Vus, B., and Gonchar, M., 2007b, Amperometric biosensors for formaldehyde assay, Proceedings of Scientific Society Named after Shevchenko. 19: 216-224.Google Scholar
  9. Dijken, van, J.P., Oostra-Demkes, G.J., Otto, R., and Harder, W., 1976, S-Formylglutathione: the substrate for formate dehydrogenase in methanol-utilizing yeasts, Arch Microbiol. 111 (1-2):77-83.CrossRefPubMedGoogle Scholar
  10. Ellenhorn, M.J., and Barceloux, D.G., 1988, Medical Toxicology - Diagnosis and Treatment of Human Poisoning. New York, NY: Elsevier Science Publishing Co., Inc., pp. 1002.Google Scholar
  11. Feron, V.J., Til, H.P., De Vrijer, F., Woutersen, R.A., Cassee, F.R., and Van Bladeren, P.J., 1991, Aldehydes: occurrence, carcinogenic potential, mechanism of action and risk assessment, Mutat. Res. 259: 363-385.CrossRefPubMedGoogle Scholar
  12. Geier, D.A., and Geier, M.R., 2004, Neurodevelopmental disorders following thimerosal-containing childhood immunizations: a follow-up analysis, Int. J. Toxicol. 23: 369-376.CrossRefPubMedGoogle Scholar
  13. Gerberich, H.R., and Seaman G.C., 1994, Encyclopedia of Chemical Technology. 4th edit. New York: John Wiley & Sons.11, pp. 929-951.Google Scholar
  14. Gonchar, Ɇ.V., Grabek, D., Ɉklejewich, B., Pavlishko, H.Ɇ., Shamlian, Ɉ.V., Sybirny, V.Ⱥ., Kotylak, Z., Rudke, K., Rudke K., Csoregi, E., and Sibirny, A.A., 2005, A new enzymo-chemical method for simultaneous assay of methanol and formaldehyde, Ukr. Biochem. J. 77 (3):146-154.Google Scholar
  15. Hinnen, A., Hicks, J.B. and Fink, G.R., 1978, Transformation of yeast, PNAS. 75: 1929-1933.CrossRefPubMedGoogle Scholar
  16. Ho, M.H., and Richards, R.A., 1990, Enzymatic method for the determination of formalde-hyde, Environ. Sci. Technol. 24: 201-204.CrossRefGoogle Scholar
  17. Ito, H., Fukuda, Y., Murata, K., and Kimura, A., 1983, Transformation of intact yeast cells treated with alkali cations, J. Bacteriol. 153(1): 163-168.PubMedGoogle Scholar
  18. Lee, B., Yurimoto, H., Sakai, Y., and Kato, N., 2002, Physiological role of the glutathione-dependent formaldehyde dehydrogenase in the methylotrophic yeast Candida boidinii, Microbiology. 148( 9): 2697-2704.PubMedGoogle Scholar
  19. Liteplo, R.G., Beauchamp, R., Meek, M.E., and Chenier, R., 2002, Formaldehyde, Geneva. www. Formaldehyde. org/who.pdf .
  20. Maidan, M.M., Gonchar, M.V., and Sibirny, A.A., 1997, Oxidation of exogenious formaldehyde in cells of methylotrophic and nonmethylotrophic yeast, Biochemistry. 62 (6): 744-749 (in Russian).Google Scholar
  21. Melissis, S.C., Rigden, D.J., Clonisa, Y.D., 2001, New family of glutathionyl-biomimetic ligands for affinity chromatography of glutathione-recognising enzymes, J. of Chroma-tography A. 917: 29-42.CrossRefGoogle Scholar
  22. National Research Council, Formaldehyde and Other Aldehydes pp. 2-1 to 5-96 USEPA 600/6-82-002 (1982).Google Scholar
  23. Ngounou, B., Neugebauer, S., Frodl, A.., Reiter, S., and Schuhmann, W., 2004, Combi-natorial synthesis of a library of acrylic acid-based polymers and their evaluation as immobilisation matrix for amperometric biosensors, Electrochim. Acta. 49: 3855-3863.CrossRefGoogle Scholar
  24. Nikitina, O., Shleev, S., Gayda, G., Demkiv, O., Gonchar, M., Gorton, L., Csöregi, E., Nistor, M., 2007, Bi-enzyme biosensor based on NAD+- and glutathione-dependent re-combinant formaldehyde dehydrogenase and diaphorase for formaldehyde assay, Sensors and Actuators B. 125:1-9.CrossRefGoogle Scholar
  25. Offit, P., and Jew, R., 2007, Addressing parents’ concerns: do vaccines contain harmful preservatives, adjuvants, additives, or residuals, Pediatrics. 112(6): 1394-1401.Google Scholar
  26. Paryzhak, S., Demkiv, O., Gayda, G., Sibirny, V., Schuhmann, W., and Gonchar, M., 2007, Enzyme- and cells-based biosensors for assay of formaldehyde in vaccines, Proc. 2nd Polish-Ukrainian Weigl Conference “Microbiology in the XXI century” (Warsow, 24-26 September, 2007). pp. 170-173.Google Scholar
  27. Patent ʋ 1213358 EP, Publ. 12.06.2002. K. Takahide, S. Atsushi, H. Shizuo et al. (JP). Method and kit for the determination of a biological component.Google Scholar
  28. Pavlishko, H.M, Honchar, T.M., and Gonchar, M.V., 2003, Chemical and enzymatic assay of formaldehyde content in fish food, Experiment. Clin. Physiol Biochem. 4:56-63 (in Ukrainian).Google Scholar
  29. Polska Norma PN-71 C-04568., 1988 Water and Waste Water. Determination of methyl alcohol content. Ed. 5 (Polish Com. Standard. eds.).Google Scholar
  30. Rehbein, H., 1995, Formaldehyd und Dimethylamin in tiefgekühlten Fischerzeugnissen aus dem Handel - eine Bestandsaufnahme, Archiv Lebensmittelhyg. 46: 122-124.Google Scholar
  31. Sawicki, E., Hauser, T.R., Stanley, T.W., and Elbert, W., 1961, The 3-methyl-2-benzothiazolinone hydrazone test. Sensitive new methods for the detection, rapid estimation, and determination of aliphatic aldehydes, Anal. Chem. 33: 93-96.CrossRefGoogle Scholar
  32. Schechter, D.S., and Singer, P.C., 1995, Formation of aldehydes during ozonation, Ozone Sci. Eng. 17: 53-55.CrossRefGoogle Scholar
  33. Schutte, H., Flossorf, J., Sahm, H., and Kula, M.R., 1976, Purification and properties of formaldehyde dehydrogenase and formate dehydrogenase from Candida boidinii, Eur. J. Biochem. 62(1): 151-160.CrossRefGoogle Scholar
  34. Sigma-Aldrich Catalogue, 2007,
  35. Smutok, O., Ngounou, B., Pavlishko, H., Gayda, G., Gonchar, M., and Schuhmann W., 2006, A reagentless bienzyme amperometric biosensor based on alcohol oxidase/peroxidase and an Os-complex modified electrodeposition paint. Sensors and Actuators B: Chemical. 113: 590-598.CrossRefGoogle Scholar

Copyright information

© Springer Science + Business Media B.V. 2008

Authors and Affiliations

  • Galyna Gayda
    • 1
  • Olha Demkiv
    • 1
  • Mykhailo Gonchar
    • 1
  • Solomiya Paryzhak
    • 2
  • Wolfgang Schuhmann
    • 3
  1. 1.Institute of Cell BiologyNAS of UkraineLvivUkraine
  2. 2.Ivan Franko National University of LvivLvivUkraine
  3. 3.Ruhr-UniversitätBochumGermany

Personalised recommendations