Galina Gayda

Overexpression of pyruvate decarboxylase in the yeast Hansenula polymorpha results in increased ethanol yield in high‐temperature fermentation of xylose

Improvement of xylose fermentation is of great importance to the fuel ethanol industry. The nonconventional thermotolerant yeast Hansenula polymorpha naturally ferments xylose to ethanol at high temperatures (48-50 degrees C). Introduction of a mutation that impairs ethanol reutilization in H. polymorpha led to an increase in ethanol yield from xylose. The native and heterologous (Kluyveromyces lactis) PDC1 genes coding for pyruvate decarboxylase were expressed at high levels in H. polymorpha under the control of the strong constitutive promoter of the glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH). This resulted in increased pyruvate decarboxylase activity and improved ethanol production from xylose. The introduction of multiple copies of the H. polymorpha PDC1 gene driven by the strong constitutive promoter led to a 20-fold increase in pyruvate decarboxylase activity and up to a threefold elevation of ethanol production.

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.

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.

The chromate resistance phenotype of some yeast mutants correlates with a lower level of Cr(V)-species generated in the extra-cellular medium

The paper describes the selection of chromate-resistant mutants of the yeast Pichia guilliermondii with a higher chromate-reducing activity and reports the EPR-study of Cr(V)-generation in the extra-cellular medium during the reduction of chromate by the yeast culture. It is shown that the reduction of chromate to Cr(III) species runs through the extra-cellular generation of Cr(V)-intermediate(s), thus supporting the assumption about the existence of an extra-cellular pathway of Cr(VI)-reduction. Furthermore, it is demonstrated that the chromate-resistance phenotype of tested mutants correlates with a lower stationary level of Cr(V)-species in the medium. It is thus suggested that isolated mutants can be used as sources of Cr(III)-biocomplexes due to their ability to effectively reduce chromate to Cr(III)-chelates with potential pharmacological applications.

Yeast-Based Biosensors for Clinical Diagnostics and Food Control

Science; medicine; clinical diagnostics; biotechnologies, including those in food and beverage industries; as well as environmental technologies need highly selective, sensitive, rapid, and reliable methods of identifying the key ingredients or metabolites which determine the quality of the product or serve as markers for diseases, the physiological state of human organism, or environmental safety. Biosensors are the most promising tool for these aims. Although the most of created biosensors are based on using enzymes as biocatalytic elements, cell sensors, especially microbial ones, have been actively developed only in recent years. A microbial biosensor consists of a transducer in conjunction with immobilized viable or nonviable microbial cells, an economical substitute for enzymes. The target analyte is usually either a substrate or an inhibitor of cell metabolism. In this review, the main achievements in the elaboration of microbial sensors, based on yeast cells, are described, and perspectives of their usage in clinical diagnostics and food control are discussed.

Amperometric Biosensors for Lactate, Alcohols, and Glycerol Assays in Clinical Diagnostics

Biosensors are bioanalytical devices which transform a biorecognition response into a measurable physical signal. Although biosensors are a novel achievement of bioanalytical chemistry, they are not only a subject of intensive research, but also a real commercial product (Kissinger, 2005). The estimated world analytical market is about

Highly selective apo-arginase based method for sensitive enzymatic assay of manganese (II) and cobalt (II) ions

20 billion per year of which 30 % is in the healthcare field. The biosensors market is expected to grow from

Yeast-Based Systems for Environmental Control

6.72 billion in 2009 to

ARGINASE-BASED AMPEROMETRIC BIOSENSOR FOR MANGANESE IONS ANALYSIS

14.42 billion in 2016 (http://www.marketresearch.com, Analytical Review of World Biosensors Market). Although up to now IUPAC has not accepted an official definition of the term biosensor, its electrochemical representative is defined as “a self-contained integrated device, which is capable of providing specifc quantitative or semi-quantitative analytical information using a biological recognition element (biochemical receptor) which is retained in direct spatial contact with an electrochemical transduction element’’ (Thevenot et al., 2001). Generally, the biosensor is a hybrid device containing two functional parts: a bioelement (an immobilized biologically active material) and a physical transducer. As bioelements pieces of tissue, microbial cells, organelles, natural biomembranes or liposomes, receptors, enzymes, antibodies and antigens, abzymes, nucleic acids and other biomolecules and even biomimetics which imitate structural and functional features of the natural analogue can be used. The bioelement is a recognition unit providing selective binding or biochemical/metabolic conversion of the analyte that result in changes in physical or physico-chemical characteristics of the transducer (Scheller et al., 1991; Schmidt & Karube, 1998; Gonchar et al., 2002; Nakamura & Karube, 2003; Sharma et al., 2003; Investigations on Sensor Systems and Technologies, 2006). The bioelement in such constructions is usually prepared in immobilized form and often covered with an outer membrane (or placed between two membranes in a sandwich manner), which either prevents the penetration of