Olha Demkiv

Immobilized formaldehyde-metabolizing enzymes from Hansenula polymorpha for removal and control of airborne formaldehyde

Formaldehyde (FA)-containing indoor air has a negative effect on human health and should be removed by intensive ventilation or by catalytic conversion to non-toxic products. FA can be oxidized by alcohol oxidase (AOX) taking part in methanol metabolism of methylotrophic yeasts. In the present work, AOX isolated from a Hansenula polymorpha C-105 mutant (gcr1 catX) overproducing this enzyme in glucose medium, was tested for its ability to oxidize airborne FA. A continuous fluidized bed bioreactor (FBBR) was designed to enable an effective bioconversion of airborne FA by AOX or by permeabilized mutant H. polymorpha C-105 cells immobilized in calcium alginate beads. The immobilized AOX having a specific activity of 6-8 U mg⁻¹ protein was shown to preserve 85-90% of the initial activity. The catalytic parameters of the immobilized enzyme were practically the same as for the free enzyme (k(cat)/K(m) was 2.35×10³ M⁻¹ s⁻¹ vs 2.89×10³ M⁻¹ s⁻¹, respectively). The results showed that upon bubbling of air containing from 0.3 up to 18.5 ppm FA through immobilized AOX in the range of 1.3-26.6 U g⁻¹ of the gel resulted in essential decrease of FA concentration in the outlet gas phase (less than 0.02-0.03 ppm, i.e. 10-fold less than the threshold limit value). It was also demonstrated that a FBBR with immobilized permeabilized C-105 cells provided more than 90% elimination of airborne FA. The process was monitored by a specially constructed enzymatic amperometric biosensor based on FA oxidation by NAD+ and glutathione-dependent formaldehyde dehydrogenase from the recombinant H. polymorpha Tf 11-6 strain.

Alcohol oxidase- and formaldehyde dehydrogenase-based enzymatic methods for formaldehyde assay in fish food products

For Gadoid fishes, formaldehyde can be generated in tissues in huge amounts during endogenous enzymatic degradation of natural osmoprotectant trimethylamine-N-oxide. This paper describes two enzymatic methods for assay of formaldehyde in fish food products using alcohol oxidase (AOX) and formaldehyde dehydrogenase (FdDH) isolated from the thermotolerant methylotrophic yeast Hansenula polymorpha. AOX-based method exploits an ability of the enzyme to oxidise a hydrated form of formaldehyde to formic acid and hydrogen peroxide monitored in peroxidase-catalysed colorimetric reaction. In FdDH-based method, a monitored coloured formazane is formed from nitrotetrazolium salt during reduction by NADH, produced in formaldehyde-dependent reaction. It was demonstrated an applicability of both methods for assay of formaldehyde in fish products. The optimal protocols for analysis procedures have been elaborated and analytical parameters of both enzymatic methods have been established. The both methods were demonstrated that some fish products (hake and cod) contain high formaldehyde concentrations (up to 100mg/kg wet weight).

Intact and permeabilized cells of the yeast Hansenula polymorpha as bioselective elements for amperometric assay of formaldehyde

Intact and permeabilized yeast cells were tested as the biorecognition elements for amperometric assay of formaldehyde (FA). For this aim, the mutant C-105 (gcr1 catX) of the methylotrophic yeast Hansenula polymorpha with a high activity of AOX was chosen. Different approaches were used for monitoring FA-dependent cell response including analysis of their oxygen consumption rate by the use of a Clark electrode, as well as assay of oxidation of redox mediator at a screen-printed platinum electrode covered by cells entrapped in Ca-alginate gel. It was shown that oxygen consumption rate of permeabilized cells reached its saturation at 4mM of FA (23 degrees C). The detection limit was found to be 0.27mM. In the presence of redox mediator 2,6-dichlorophenolindophenol (DCIP), the screen-printed platinum band electrode covered by permeabilized cells did not show any current output to FA. In contrast, well-pronounced amperometric response to FA was observed in the case of intact yeast cells in the presence of DCIP. It was shown that current output reached its maximum at 7mM concentration of FA. The detection limit was found to be 0.74mM. Obviously, it is necessary to perform a directed genetic engineering of the yeast cells to improve their bioanalytical characteristics in the corresponding biosensors.

Formaldehyde Oxidizing Enzymes and Genetically Modified Yeast Hansenula polymorpha Cells in Monitoring and Removal of Formaldehyde

Vladimir Sibirny2, Olha Demkiv1, Sasi Sigawi4,5, Solomiya Paryzhak1, Halyna Klepach1, Yaroslav Korpan3, Oleh Smutok1, Marina Nisnevich4, Galina Gayda1, Yeshayahu Nitzan5, Czeslaw Puchalski2 and Mykhailo Gonchar1,2 1Institute of Cell Biology NAS of Ukraine, Lviv, 2University of Rzeszow, Rzeszow-Kolbuszowa, 3Institute of Molecular Biology & Genetics NAS of Ukraine, Kyiv, 4Ariel University Center of Samaria, Ariel, 5Bar-Ilan University, Ramat-Gan, 1,3Ukraine 2Poland 4,5Israel

Detection of Waterborne and Airborne Formaldehyde: From Amperometric Chemosensing to a Visual Biosensor Based on Alcohol Oxidase

A laboratory prototype of a microcomputer-based analyzer was developed for quantitative determination of formaldehyde in liquid samples, based on catalytic chemosensing elements. It was shown that selectivity for the target analyte could be increased by modulating the working electrode potential. Analytical parameters of three variants of the amperometric analyzer that differed in the chemical structure/configuration of the working electrode were studied. The constructed analyzer was tested on wastewater solutions that contained formaldehyde. A simple low-cost biosensor was developed for semi-quantitative detection of airborne formaldehyde in concentrations exceeding the threshold level. This biosensor is based on a change in the color of a solution that contains a mixture of alcohol oxidase from the yeast Hansenula polymorpha, horseradish peroxidase and a chromogen, following exposure to airborne formaldehyde. The solution is enclosed within a membrane device, which is permeable to formaldehyde vapors. The most efficient and sensitive biosensor for detecting formaldehyde was the one that contained alcohol oxidase with an activity of 1.2 U·mL−1. The biosensor requires no special instrumentation and enables rapid visual detection of airborne formaldehyde at concentrations, which are hazardous to human health.

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

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.

A Reagentless Amperometric Formaldehyde-Selective Chemosensor Based on Platinized Gold Electrodes

Fabrication and characterization of a new amperometric chemosensor for accurate formaldehyde analysis based on platinized gold electrodes is described. The platinization process was performed electrochemically on the surface of 4 mm gold planar electrodes by both electrolysis and cyclic voltamperometry. The produced electrodes were characterized using scanning electron microscopy and X-ray spectral analysis. Using a low working potential (0.0 V vs. Ag/AgCl) enabled an essential increase in the chemosensor’s selectivity for the target analyte. The sensitivity of the best chemosensor prototype to formaldehyde is uniquely high (28180 A·M−1·m−2) with a detection limit of 0.05 mM. The chemosensor remained stable over a one-year storage period. The formaldehye-selective chemosensor was tested on samples of commercial preparations. A high correlation was demonstrated between the results obtained by the proposed chemosensor, chemical and enzymatic methods (R = 0.998). The developed formaldehyde-selective amperometric chemosensor is very promising for use in industry and research, as well as for environmental control.