Demkiv O. Formaldehyde dehydrogenase of recombinant strains Нansenula polymorpha: optimization of synthesis, purification and application in biosensor's and enzymatic assay of formaldehyde

Object of the research: formaldehyde detoxification with recombinant yeast strains H. polymorpha, resistant to elevated formaldehyde (FA) concentrations, and development of enzymatic methods for FA assay based on formaldehyde dehydrogenase (FdDH). The aim of the work: to screen the gene-engineered strains of the yeast H. рolymorpha - over-producers of NAD+- and glutathione-dependent FdDH; to study the details of regulation of FdDH productivity in yeast cells under different cultivation conditions at elevated FA content; to develop the simple scheme for FdDH isolation and purification; to create the new methods FdDH-based for FA testing. Methods: microbiological, genetic, biochemical, mathematical statistics. The number of wild and gene-engineered strains of yeast H. рolymorpha were screened and the stable recombinant strain - over-producer of FdDH, possessing elevated resistance to FA (up to 15 mM), was chosen at the first time. The details of FdDH synthesis and other's enzymes of FA catabolism in recombinant strains were studied and cultivation conditions for maximal FdDH yield were optimized. It was shown that FdDH activity is dependent on the carbon source and FA content in cultural medium, FA is an inducer of FdDH synthesis. The described results lead us to understanding the detailed mechanisms of FA detoxification in methylotrophic yeast, especially at high content of FA in growth medium.The recombinant strains H. polymorpha, being resistant to FA, were used as model to investigate adaptation ability of cells to live under elevated FA levels. Then, these recombinants were used as a FA- oxidizing cartridge in bioreactor for biodegradation of FA in air and liquid. The simple scheme for FdDH isolation and purification from the gene-engineered strain H. рolymorpha was firstly proposed and the FdDH-based reagentless biosensor to monitor FA was constructed. FdDH was used for the development of enzymatic spectrophotometric assay of FA in water wastes. This method can be recommended for the development of state standard of FA analyses and the corresponding commercial analytical test-kit. Fabricated laboratory prototype of biosensor was used for construction of experimental sample of FA analyser. The methods of FA assay, described in this study, may be utilized in practice - in laboratories of ecology, sanitary, factories, medico-diagnostics and scientific research. Area of use: microbiology, biochemistry.