"Green" Prussian Blue Analogues as Peroxidase Mimetics for Amperometric Sensing and Biosensing.

Prussian blue analogs (PBAs) are well-known artificial enzymes with peroxidase (PO)-like activity. PBAs have a high potential for applications in scientific investigations, industry, ecology and medicine. Being stable and both catalytically and electrochemically active, PBAs are promising in the construction of biosensors and biofuel cells. The "green" synthesis of PO-like PBAs using oxido-reductase flavocytochrome b2 is described in this study. When immobilized on graphite electrodes (GEs), the obtained green-synthesized PBAs or hexacyanoferrates (gHCFs) of transition and noble metals produced amperometric signals in response to H2O2. HCFs of copper, iron, palladium and other metals were synthesized and characterized by structure, size, catalytic properties and electro-mediator activities. The gCuHCF, as the most effective PO mimetic with a flower-like micro/nano superstructure, was used as an H2O2-sensitive platform for the development of a glucose oxidase (GO)-based biosensor. The GO/gCuHCF/GE biosensor exhibited high sensitivity (710 A M-1m-2), a broad linear range and good selectivity when tested on real samples of fruit juices. We propose that the gCuHCF and other gHCFs synthesized via enzymes may be used as artificial POs in amperometric oxidase-based (bio)sensors.

Alcohol Oxidase from the Methylotrophic Yeast Ogataea polymorpha: Isolation, Purification, and Bioanalytical Application.

Alcohol oxidase (EC 1.1.3.13; AOX) is a flavoprotein that catalyzes the oxidation of primary short-chain alcohols to corresponding carbonyl compounds with a concomitant release of hydrogen peroxide. It is a key enzyme of methanol metabolism in methylotrophic yeasts, catalyzing the first step of methanol oxidation to formaldehyde.Here we describe the isolation and purification of AOX from the thermotolerant methylotrophic yeast Ogataea (Hansenula) polymorpha, and using this enzyme in enzymatic assay of ethanol, simultaneous analysis of methanol and formaldehyde, and in construction of amperometric biosensors selective to primary alcohols and formaldehyde.

Extracellular laccase from Monilinia fructicola: isolation, primary characterization and application.

Laccases are enzymes belonging to the family of blue copper oxidases. Due to their broad substrate specificity, they are widely used in many industrial processes and environmental bioremediations for removal of a large number of pollutants. During last decades, laccases attracted scientific interest also as highly promising enzymes to be used in bioanalytics. The aim of this study is to obtain a highly purified laccase from an efficient fungal producer and to demonstrate the applicability of this enzyme for analytics and bioremediation. To select the best microbial source of laccase, a screening of fungal strains was carried out and the fungus Monilinia fructicola was chosen as a producer of an extracellular enzyme. Optimal cultivation conditions for the highest yield of laccase were established; the enzyme was purified by a column chromatography and partially characterized. Molecular mass of the laccase subunit was determined to be near 35 kDa; the optimal pH ranges for the highest activity and stability are 4.5-5.0 and 3.0-5.0, respectively; the optimal temperature for laccase activity is 30°C. Laccase preparation was successfully used as a biocatalyst in the amperometric biosensor for bisphenol A assay and in the bioreactor for bioremediation of some xenobiotics.

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.

Formaldehyde dehydrogenase from the recombinant yeast Hansenula polymorpha: isolation and bioanalytic application.

A recombinant yeast clone, a derivative of the recipient Hansenula polymorpha strain NCYC 495, was chosen as an NAD and glutathione-dependent formaldehyde dehydrogenase overproducer. Optimal cultivation conditions for the highest yield of enzyme were established. A simple scheme for the isolation of formaldehyde dehydrogenase from the recombinant strain was proposed, and some characteristics of the purified enzyme were studied. An enzymatic method for formaldehyde assay based on formaldehyde dehydrogenase was developed and used for testing real samples.