Intact Yeast Research Articles

Imaging-Based Live Cell Yeast Screen Identifies Novel Factors Involved in Peroxisome Assembly

We describe an imaging-based method in intact cells to systematically screen yeast mutant libraries for abnormal morphology and distribution of fluorescently labeled subcellular structures. In this study, chromosomally expressed green fluorescent protein (GFP) fused to the peroxisomal targeting sequence 1, consisting of serine-lysine-leucine, was introduced into 4740 viable yeast deletion mutants using a modified synthetic genetic array (SGA) technology. A benchtop robot was used to create ordered high-density arrays of GFP-expressing yeast mutants on solid media plates. Immobilized live yeast colonies were subjected to high-resolution, multidimensional confocal imaging. A software tool was designed for automated processing and quantitative analysis of acquired multichannel three-dimensional image data. The study resulted in the identification of two novel proteins, as well as of all previously known proteins required for import of proteins bearing peroxisomal targeting signal PTS1, into yeast peroxisomes. The modular method enables reliable microscopic analysis of live yeast mutant libraries in a universally applicable format on standard microscope slides, and provides a step toward fully automated high-resolution imaging of intact yeast cells.

Pre-tRNA turnover catalyzed by the yeast nuclear RNase P holoenzyme is limited by product release

Ribonuclease P (RNase P) is a ribonucleoprotein that catalyzes the 5' maturation of precursor transfer RNA in the presence of magnesium ions. The bacterial RNase P holoenzyme consists of one catalytically active RNA component and a single essential but catalytically inactive protein. In contrast, yeast nuclear RNase P is more complex with one RNA subunit and nine protein subunits. We have devised an affinity purification protocol to gently and rapidly purify intact yeast nuclear RNase P holoenzyme for transient kinetic studies. In pre-steady-state kinetic studies under saturating substrate concentrations, we observed an initial burst of tRNA formation followed by a slower, linear, steady-state turnover, with the burst amplitude equal to the concentration of the holoenzyme used in the reaction. These data indicate that the rate-limiting step in turnover occurs after pre-tRNA cleavage, such as mature tRNA release. Additionally, the steady-state rate constants demonstrate a large dependence on temperature that results in nonlinear Arrhenius plots, suggesting that a kinetically important conformational change occurs during catalysis. Finally, deletion of the 3' trailer in pre-tRNA has little or no effect on the steady-state kinetic rate constants. These data suggest that, despite marked differences in subunit composition, the minimal kinetic mechanism for cleavage of pre-tRNA catalyzed by yeast nuclear RNase P holoenzyme is similar to that of the bacterial RNase P holoenzyme.

Location of Subunit d in the Peripheral Stalk of the ATP Synthase from Saccharomyces cerevisiae

ATP synthase from Saccharomyces cerevisiae is an approximately 600 kDa membrane protein complex. The enzyme couples the proton motive force across the mitochondrial inner membrane to the synthesis of ATP from ADP and inorganic phosphate. The peripheral stalk subcomplex acts as a stator, preventing the rotation of the soluble F 1 region relative to the membrane-bound F O region during ATP synthesis. Component subunits of the peripheral stalk are Atp5p (OSCP), Atp4p (subunit b), Atp7p (subunit d), and Atp14p (subunit h). X-ray crystallography has defined the structure of a large fragment of the bovine peripheral stalk, including 75% of subunit d (residues 3-123). Docking the peripheral stalk structure into a cryo-EM map of intact yeast ATP synthase showed that residue 123 of subunit d lies close to the bottom edge of F 1. The 37 missing C-terminal residues are predicted to either fold back toward the apex of F 1 or extend toward the membrane. To locate the C terminus of subunit d within the peripheral stalk of ATP synthase from S. cerevisiae, a biotinylation signal was fused to the protein. The biotin acceptor domain became biotinylated in vivo and was subsequently labeled with avidin in vitro. Electron microscopy of the avidin-labeled complex showed the label tethered close to the membrane surface. We propose that the C-terminal region of subunit d spans the gap from F 1 to F O, reinforcing this section of the peripheral stalk.

Surface Energetics to Assess Microbial Adhesion onto Fluidized Chromatography Adsorbents

AbstractCell‐to‐support interaction and cell‐to‐cell aggregation phenomena have been studied in a model system composed of intact yeast cells and agarose‐based chromatography adsorbent surfaces. Biomass components and beaded adsorbents were characterized by contact angle determinations with three diagnostic liquids and, complementarily, by zeta potential measurements. Such experimental characterization of the interacting surfaces has allowed the calculation of interfacial free energy of interaction in aqueous media vs. distance profiles. The extent of biomass adhesion was inferred from calculations performed assuming standard chromatographic conditions, but different adsorption modes. Several stationary support/mobile phase systems were considered, i.e., ion exchange, hydrophobic interaction, and pseudo‐affinity. The calculated interaction energy minima revealed marginal attraction between cells and cation exchangers or agarose‐matrix beads (U ≤ |10–20| kT) but strong attraction with anion exchangers (U ≥ |200–1000| kT). Other systems including hydrophobic interaction and chelating beads showed intermediate energy minimum values (U <$>\approx<$> |40–100| kT) for interaction with biological particles. However, the calculations also showed that working conditions in the presence of salt can promote cell aggregation apart from cell‐to‐support interaction. Predictions based on the application of the XDLVO approach were confirmed by independent experimental methods, e.g., biomass deposition experiments and laser diffraction spectroscopy. The understanding of biomass attachment onto chromatographic supports can help in alleviating process limitations normally encountered during direct (primary) sequestration of bioproducts.

MALDI-TOF mass signatures for differentiation of yeast species, strain grouping and monitoring of morphogenesis markers

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is demonstrated to be a potentially useful tool for the rapid identification of yeasts, the grouping of Candida albicans strains, and the monitoring of germ tube-specific markers. Co-crystallized with sinapinic acid as the MALDI matrix, intact yeast cells yielded a sufficient number of medium-sized ions (4-15 kDa) in MALDI mass spectra to provide "mass signatures" that were diagnostic of strain type. For most isolates, the mass signatures were affected by the growth medium, length of incubation and the cell preparation method. While the overall past success of this methodology for fungal cells has been relatively low compared to its application to bacteria, fixing the yeast cells in 50% methanol inactivated the cells, reduced cell aggregation in aqueous suspension solution, and more importantly, it significantly improved the mass signature quality. This simple but critical advance in sample treatment improved mass spectrometric signal-to-noise ratios and allowed the identification of yeasts by a mass signature approach. Under optimized conditions, Candida species (C. albicans, C. glabrata, C. krusei, C. kefyr), Aspergillus species (A. terreus, A. fumigatus, A. syndowii) and other yeast genera (Cryptococcus neoformans, Saccharomyces cerevisiae and a Rhodotorula sp.) could be distinguished. Within the C. albicans species, several common ions in the m/z 5,000-10,000 range were apparent in the mass spectra of all tested strains. In addition to shared ions, the mass spectra of individual C. albicans strains permitted grouping of the strains. Principal component analysis (PCA) was employed to confirm spectral reproducibility and C. albicans strain grouping by mass signatures. Finally, C. albicans germ tubes produced MALDI-TOF mass signatures that differed from yeast forms of this species. This is a rapid, sensitive and simple method for identifying yeasts, grouping strains and following the morphogenesis of C. albicans.

Surface energetics to assess biomass attachment onto hydrophobic interaction adsorbents in expanded beds

Cell-to-support interaction and cell-to-cell aggregation phenomena have been studied in a model system composed of intact yeast cells and Phenyl-Streamline adsorbents. Biomass components and beaded adsorbents were characterized by contact angle determinations with three diagnostic liquids and zeta potential measurements. Subsequently, free energy of interaction vs. distance profiles between interacting surfaces was calculated in the aqueous media provided by operating mobile phases. The effect of pH and ammonium sulphate concentration within the normal operating ranges was evaluated. Calculation indicated that moderate interaction between cell particles and adsorbent beads can develop in the presence of salt. Cell-to-cell aggregation was suspected to occur at high salt concentration and neutral pH. Predictions based on the application of the XDLVO approach were confirmed by independent experimental methods like biomass deposition experiments and laser diffraction spectroscopy. Understanding biomass attachment onto hydrophobic supports can help in alleviating process limitations normally encountered during expanded bed adsorption of bioproducts.

Visualized investigation of yeast transformation induced with Li + and polyethylene glycol

The effects of Li + and polyethylene glycol (PEG) on the genetic transformation of Saccharomyces cerevisiae were investigated by using fluorescence microscopy (FM) to visualize the binding of plasmid DNA labeled with YOYO-1 to the surface of yeast cells, scanning electron microscopy (SEM) and atomic force microscopy (AFM) to image the change in surface topography of yeast cells, coupled with transformation frequency experiments. The results showed that under the same conditions, the transformation frequencies of yeast protoplasts were much higher than those of intact yeast cells. PEG was absolutely required for the binding of DNA to the surface of intact yeast cells or yeast protoplasts, and had no effect on the surface topography of intact yeast cells or yeast protoplasts. In the presence of PEG, Li + could greatly enhance the binding of plasmid DNA to the surface of intact yeast cells, increase their transformation frequency, and affect their surface topography. On the other hand, no effect on the DNA binding to the surface of protoplasts and no increase in the number of transformants and no surface topography changes were found upon the treatment with Li + to protoplasts. In the present work, the effects of Li + and PEG on yeast genetic transformation were directly visualized, rather than those deduced from the results of transformation frequencies. These results indicate that cell wall might be a barrier for the uptake of plasmid DNA. Li + could increase the permeability of yeast cell wall, then increase the exposed sites of DNA binding on intact yeast cells. The main role of PEG was to induce DNA binding to cell surface.

Investigating the interaction mechanism between zinc and Saccharomyces cerevisiae using combined SEM-EDX and XAFS

The interaction mechanism between zinc and the intact yeast cells of Saccharomyces cerevisiae was investigated by using the scanning electron microscopy with energy-dispersive X-ray analysis, as well as X-ray absorption fine structure spectroscopy (XAFS). Displacement of H+, K+, Mg2+, and Na+ during zinc uptake confirmed the existence of both covalent interactions and ionic interactions between Zn2+ and the microbe. Ion exchange mechanism played a role in zinc uptake. The local environment of Zn accumulated in the intact yeast cells was determined by XAFS, which suggests that the nearest neighboring atom of the bound zinc ion on the biomass is oxygen atom. The adsorbed zinc ion on the intact cells of S. cerevisiae is a tetrahedron structure, with the Zn-O bond length of 1.97 A, and the coordination number is only 3.2 of Zn-O structure in the first shell.

X-ray Structures of U2 snRNA−Branchpoint Duplexes Containing Conserved Pseudouridines

A pseudouridine-modified region of the U2 small nuclear (sn)RNA anneals with the intronic branchpoint sequence and positions a bulged adenosine to serve as the nucleophile in the first chemical step of pre-mRNA splicing. We have determined three X-ray structures of RNA oligonucleotides containing the pseudouridylated U2 snRNA and the branchpoint consensus sequences. The expected adenosine branchpoint is extrahelical in a 1.65 A resolution structure containing the mammalian consensus sequence variant and in a 2.10 A resolution structure containing a shortened Saccharomyces cerevisiae consensus sequence. The adenosine adjacent to the expected branchpoint is extrahelical in a third structure, which contains the intact yeast consensus sequence at 1.57 A resolution. The hydration and base stacking interactions mediated by the U2 snRNA pseudouridines correlate with the identity of the unpaired adenosine. The expected adenosine bulge is associated with a well-stacked pseudouridine, which is linked via an ordered water molecule to a neighboring nucleotide. In contrast, the bulge of the adjacent adenosine shifts the base stacking and disrupts the water-mediated interactions of the pseudouridine. These structural differences may contribute to the ability of the pseudouridine modification to promote the bulged conformation of the branch site adenosine and to enhance catalysis by snRNAs. Furthermore, iodide binding sites are identified adjacent to the unconventional bulged adenosine, and the structure of the mammalian consensus sequence variant provides a high-resolution view of a hydrated magnesium ion bound in a similar manner to a divalent cation binding site of the group II intron.

Stoichiometry of the Peripheral Stalk Subunits E and G of Yeast V1-ATPase Determined by Mass Spectrometry

The stoichiometry of yeast V(1)-ATPase peripheral stalk subunits E and G was determined by two independent approaches using mass spectrometry (MS). First, the subunit ratio was inferred from measuring the molecular mass of the intact V(1)-ATPase complex and each of the individual protein components, using native electrospray ionization-MS. The major observed intact complex had a mass of 593,600 Da, with minor components displaying masses of 553,550 and 428,300 Da, respectively. Second, defined amounts of V(1)-ATPase purified from yeast grown on (14)N-containing medium were titrated with defined amounts of (15)N-labeled E and G subunits as internal standards. Following protease digestion of subunit bands, (14)N- and (15)N-containing peptide pairs were used for quantification of subunit stoichiometry using matrix-assisted laser desorption/ionization-time of flight MS. Results from both approaches are in excellent agreement and reveal that the subunit composition of yeast V(1)-ATPase is A(3)B(3)DE(3)FG(3)H.

Colloid deposition experiments as a diagnostic tool for biomass attachment onto bioproduct adsorbent surfaces

AbstractBACKGROUND: Detrimental processing conditions can be expected in any downstream operation where direct contacting between a crude feedstock and a reactive solid phase is supposed to occur. In this paper we have investigated the factors influencing intact yeast cells deposition onto anion and cation exchangers currently utilized for expanded‐bed adsorption of biotechnological products. The aim of this study was twofold: (a) to confirm previous findings relating biomass deposition with surface energetics according to the extended Derjaguin, Landau, Verwey and Overbeek theory (XDLVO) theory; and (b) to provide a simple experimental tool to evaluate biomass deposition onto process surfaces.RESULTS:Biomass deposition experiments were performed on an automated workstation utilizing a packed‐bed format. Two commercial ion exchangers intended for the direct capture of bioproducts in the presence of suspended biological particles were employed. Intact yeast cells in the late exponential phase of growth were selected as model bio‐colloids. Cell deposition was systematically evaluated as a function of fluid‐phase conductivity and quantitatively expressed as a biomass deposition parameter (α).CONCLUSION: α ≤0.15 was established as a criterion to reflect negligible biomass adhesion to the process support(s). Biomass deposition experiments further confirmed predictions made on the basis of free interfacial energy calculations as per the extended DLVO approach. Copyright © 2008 Society of Chemical Industry

Assessing adsorbent–biomass interactions during expanded bed adsorption onto ion exchangers utilizing surface energetics

Biomass adhesion onto an adsorbent matrix or “interaction” as well as biological particle co-adhesion or “aggregation” can severely affect the overall performance of many direct-contact methods for downstream processing of bioproducts. Studies to quantitatively describe this biomass–adsorbent interaction were developed utilizing surface energetics. An indirect thermodynamic approach via contact angle and zeta potential measurements was utilized. Intact yeast cells, yeast homogenates, and disrupted bacterial paste were employed as model system. Various surfaces that are relevant to biochemical and environmental applications were characterized. The extended Derjaguin, Landau, Verwey, Overbeek (XDLVO) theory was found to appropriately predict biomass adhesion behaviour. It was observed that cell attachment onto anion-exchange supports is promoted by strong and close interaction within a secondary energy minimum followed by moderate multilayer cell aggregation. On the other hand, cell interaction with cation-exchange materials can take place within a reversible secondary energy minimum and at longer separation distance. The influence of particle charge and size, as well as the influence of the nature of the material under study were summarized in the form of energy vs. distance profiles. These investigations lead to many process-related conclusions: (a) process buffer conductivity windows can be recommended for anion-exchange chromatography (AEX) vs. cation-exchange chromatography (CEX) systems, (b) increased hydrodynamic shear is required to prevent biomass attachment onto AEX as compared to CEX, and (c) aggregation phenomena is a function of contact time and biomass concentration. Understanding biomass–adsorbent interaction at the particle (local) level is opening the pave for optimized operation of expanded bed adsorption methods at the process (macro) scale. A universal methodological approach is presented to guide both process and material design.

Preparation of intact yeast artificial chromosome DNA for transgenesis of mice

Transgenesis with large DNA molecules such as yeast artificial chromosomes (YACs) has an advantage over smaller constructs in that an entire locus and all its flanking cis-regulatory elements are included. The key to obtaining animals bearing full-length transgenes is to avoid physical shearing of the DNA during purification and microinjection. This protocol details how to prepare intact YAC DNA for transgenesis of mice and involves separation of YAC DNA from yeast chromosomal DNA by pulsed field gel electrophoresis, concentration to a range suitable for microinjection by second dimension electrophoresis and enzymatic digestion of matrix-embedded YAC DNA to produce a solution that can be injected. The YAC is maintained in an agarose gel matrix to avoid damage until the final steps before microinjection. Special precautions are also taken during the microinjection protocol. Transgenesis efficiency is approximately 15%; most animals carry 1-5 copies of the desired locus. This method takes 6 d for completion.

Functional Characterization of AtATM1, AtATM2, and AtATM3, a Subfamily of Arabidopsis Half-molecule ATP-binding Cassette Transporters Implicated in Iron Homeostasis

The functional capabilities of one of the smallest subfamilies of ATP-binding cassette transporters from Arabidopsis thaliana, the AtATMs, are described. Designated AtATM1, AtAATM2, and AtATM3, these half-molecule ABC proteins are homologous to the yeast mitochondrial membrane protein ATM1 (ScATM1), which is clearly implicated in the export of mitochondrially synthesized iron/sulfur clusters. Yeast ATM1-deficient (atm1) mutants grow very slowly (have a petite phenotype), are respiration-deficient, accumulate toxic levels of iron in their mitochondria, and show enhanced compensatory high affinity iron uptake. Of the three Arabidopsis ATMs, AtATM3 bears the closest functional resemblance to ScATM1. Heterologously expressed AtATM3 is not only able to complement the yeast atm1 petite phenotype but is also able to suppress the constitutively high capacity for high affinity iron uptake associated with loss of the chromosomal copy of ScATM1, abrogate intra-mitochondrial iron hyperaccumulation, and restore mitochondrial respiratory function and cytochrome c levels. By comparison, AtATM1 only weakly suppresses the atm1 phenotype, and AtATM2 exerts little or no suppressive action but instead is toxic when expressed in this system. The differences between AtATM3 and AtATM1 are maintained after exchanging their target peptides, and these proteins as well as AtATM2 colocalize with the mitochondrial fluor MitoTracker Red when expressed in yeast as GFP fusions. Although its toxicity when heterologously expressed in yeast, except when fused with GFP, precludes the functional analysis of native AtATM2, a common function, mitochondrial export of Fe/S clusters or their precursors for the assembly of cytosolic Fe/S proteins, is inferred for AtATM3 and AtATM1.

Noncovalent Binding of Small Ubiquitin-related Modifier (SUMO) Protease to SUMO Is Necessary for Enzymatic Activities and Cell Growth

SUMO proteases possess two enzymatic activities to hydrolyze the C-terminal region of SUMOs (hydrolase activity) and to remove SUMO from SUMO-conjugated substrates (isopeptidase activity). SUMO proteases bind to SUMOs noncovalently, but the physiological roles of the binding in the functions of SUMO proteases are not well understood. In this study we found that SUMO proteases (Axam, SENP1, and yeast Ulp1) show different preferences for noncovalent binding to various SUMOs (SUMO-1, -2, -3, and yeast Smt3) and that the hydrolase and isopeptidase activities of SUMO proteases are dependent on their binding to SUMOs through salt bridge. Expression of Smt3 suppressed the phenotype of yeast mutant lacking smt3, which exhibits growth arrest, and the binding of Ulp1 to Smt3 was essential for this rescue activity. Although expression of an Smt3 mutant (smt3R64E(GG)), which conjugates to substrate but loses the ability to bind to Ulp1, rescued the phenotype of yeast lacking smt3 partially, the mutant cells showed an increment in the doubling time and a delay of desumoylation of Smt3-conjugated Cdc3. These results indicate that the noncovalent binding of SUMO protease to SUMO through salt bridge is essential for the enzymatic activities and that the balance between sumoylation and desumoylation is important for cell growth control.

An ectophosphatase activity inCandida parapsilosisinfluences the interaction of fungi with epithelial cells

This study describes the biochemical characterization of a phosphatase activity present on the cell surface of Candida parapsilosis, a common cause of candidemia. Intact yeasts hydrolyzed p-nitrophenylphosphate to p-nitrophenol at a rate of 24.30+/-2.63 nmol p-nitrophenol h(-1) 10(-7) cells. The cell wall distribution of the Ca. parapsilosis enzyme was demonstrated by transmission electron microscopy. The duration of incubation of the yeast cells with the substrate and cell density influenced enzyme activity linearly. Values of V(max) and apparent K(m) for p-nitrophenylphosphate hydrolysis were 26.80+/-1.13 nmol p-nitrophenol h(-1) 10(-7) cells and 0.47+/-0.05 mM p-nitrophenylphosphate, respectively. The ectophosphatase activity was strongly inhibited at high pH as well as by classical inhibitors of acid phosphatases, such as sodium orthovanadate, sodium molybdate, sodium fluoride, and inorganic phosphate, the final product of the reaction. Only the inhibition caused by sodium orthovanadate was irreversible. Different phophorylated amino acids were used as substrates for the Ca. parapsilosis ectoenzyme, and the highest rate of phosphate hydrolysis was achieved using phosphotyrosine. A direct relationship between ectophosphatase activity and adhesion to host cells was established. In these assays, irreversible inhibition of enzyme activity resulted in decreased levels of yeast adhesion to epithelial cells.

Water Transport in Intact Yeast Cells as Assessed by Fluorescence Self-Quenching

Intact yeast cells loaded with 5- and-6-carboxyfluorescein were used to assess water transport. The results were similar to those previously reported for protoplasts assessed by using either fluorescence or light scattering, and the activation energies were 8.0 and 15.1 kcal mol(-1) (33.4 and 63.2 kJ mol(-1)) for a strain overexpressing AQY1 aquaporin and a parental strain, respectively.

Specific Aquaporins Facilitate the Diffusion of Hydrogen Peroxide across Membranes

The metabolism of aerobic organisms continuously produces reactive oxygen species. Although potentially toxic, these compounds also function in signaling. One important feature of signaling compounds is their ability to move between different compartments, e.g. to cross membranes. Here we present evidence that aquaporins can channel hydrogen peroxide (H2O2). Twenty-four aquaporins from plants and mammals were screened in five yeast strains differing in sensitivity toward oxidative stress. Expression of human AQP8 and plant Arabidopsis TIP1;1 and TIP1;2 in yeast decreased growth and survival in the presence of H2O2. Further evidence for aquaporin-mediated H2O2 diffusion was obtained by a fluorescence assay with intact yeast cells using an intracellular reactive oxygen species-sensitive fluorescent dye. Application of silver ions (Ag+), which block aquaporin-mediated water diffusion in a fast kinetics swelling assay, also reversed both the aquaporin-dependent growth repression and the H2O2-induced fluorescence. Our results present the first molecular genetic evidence for the diffusion of H2O2 through specific members of the aquaporin family.

Three mammalian cytochromes b561 are ascorbate‐dependent ferrireductases

Cytochromes b(561) are a family of transmembrane proteins found in most eukaryotic cells. Three evolutionarily closely related mammalian cytochromes b(561) (chromaffin granule cytochrome b, duodenal cytochrome b, and lysosomal cytochrome b) were expressed in a Saccharomyces cerevisiaeDeltafre1Deltafre2 mutant, which lacks almost all of its plasma membrane ferrireductase activity, to study their ability to reduce ferric iron (Fe(3+)). The expression of each of these cytochromes b(561) was able to rescue the growth defect of the Deltafre1Deltafre2 mutant cells in iron-deficient conditions, suggesting their involvement in iron metabolism. Plasma membrane ferrireductase activities were measured using intact yeast cells. Each cytochrome b(561) showed significant FeCN and Fe(3+)-EDTA reductase activities that were dependent on the presence of intracellular ascorbate. Site-directed mutagenesis of lysosomal cytochrome b was conducted to identify amino acids that are indispensable for its activity. Among more than 20 conserved or partially conserved amino acids that were investigated, mutations of four His residues (H47, H83, H117 and H156), one Tyr (Y66) and one Arg (R67) completely abrogated the FeCN reductase activity, whereas mutations of Arg (R149), Phe (F44), Ser (S115), Trp (W119), Glu (E196), and Gln (Q131) affected the ferrireductase activity to some degree. These mutations may affect the heme coordination, ascorbate binding, and/or ferric substrate binding. Possible roles of these residues in lysosomal cytochrome b are discussed. This study demonstrates the ascorbate-dependent transmembrane ferrireductase activities of members of the mammalian cytochrome b(561) family of proteins.

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 4 mM of FA (23 °C). The detection limit was found to be 0.27 mM. 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 7 mM concentration of FA. The detection limit was found to be 0.74 mM. Obviously, it is necessary to perform a directed genetic engineering of the yeast cells to improve their bioanalytical characteristics in the corresponding biosensors.