Further insights into the catalytical properties of deglycosylated pyranose dehydrogenase from Agaricus meleagris recombinantly expressed in Pichia pastoris.

The present study focuses on fragmented deglycosylated pyranose dehydrogenase (fdgPDH) from Agaricus meleagris recombinantly expressed in Pichia pastoris . Fragmented deglycosylated PDH is formed from the deglycosylated enzyme (dgPDH) when it spontaneously loses a C-terminal fragment when stored in a buffer solution at 4 °C. The remaining larger fragment has a molecular weight of ∼46 kDa and exhibits higher volumetric activity for glucose oxidation compared with the deglycosylated and glycosylated (gPDH) forms of PDH. Flow injection amperometry and cyclic voltammetry were used to assess and compare the catalytic activity of the three investigated forms of PDH, "wired" to graphite electrodes with two different osmium redox polymers: [Os(4,4'-dimethyl-2,2'-bipyridine)2(poly(vinylimidazole))10Cl](+) [Os(dmbpy)PVI] and [Os(4,4'-dimethoxy-2,2'-bipyridine)2(poly-(vinylimidazole))10Cl](+) [Os(dmobpy)PVI]. When "wired" with Os(dmbpy)PVI, the graphite electrodes modified with fdgPDH showed a pronounced increase in the current density with Jmax 13- and 6-fold higher than that observed for gPDH- and dgPDH-modified electrodes, making the fragmented enzyme extraordinarily attractive for further biotechnological applications. An easier access of the substrate to the active site and improved communication between the enzyme and mediator matrix are suggested as the two main reasons for the excellent performance of the fdgPDH when compared with that of gPDH and dgPDH. Three of the four glycosites in PDH: N(75), N(175), and N(252) were assigned using mass spectrometry in conjunction with endoglycosidase treatment and tryptic digestion. Determination of the asparagine residues carrying carbohydrate moieties in PDH can serve as a solid background for production of recombinant enzyme lacking glycosylation.

Graphite electrodes modified with Neurospora crassa cellobiose dehydrogenase: comparative electrochemical characterization under direct and mediated electron transfer.

The electrochemical characterization of a class II cellobiose dehydrogenase (CDH), from the ascomycete fungus Neurospora crassa, adsorbed on graphite (G), was performed in regard to direct (DET) and mediated electron transfer (MET). The effects of the applied potential, mediator (1,4 benzoquinone) concentration and flow carrier pH on the amperometric response of the G/CDH modified electrodes were investigated under flow conditions. From the calibration curves, recorded at two pH values (5.2 and 7.0) for nine different sugars, the kinetic and the analytical parameters were evaluated under DET and MET operation modes. These results together with those obtained from long term operational stability measurements showed that: (i) for all nine investigated sugars the sensitivity was higher for MET than for DET and for pH 5.2 compared to pH 7.0; (ii) irrespective of DET or MET operation mode, the sensitivity of the new enzyme towards the investigated sugars decreased in the following sequence: cellobiose>lactose>(cellotriose≈cellopentaose) >>(maltotriose≈maltotetraose≈maltopentaose)>(maltose≈glucose); (iii) for all tested substrates, the apparent CDH affinity was roughly higher in DET than in MET operation mode.

Displacement immunoassay for the detection of ochratoxin A using ochratoxin B modified glass beads.

We report here the development of a new assay for the detection of ochratoxin A (OTA) based on the use of its dechlorinated analogue, ochratoxin B (OTB), in a displacement immunoassay. OTB was immobilised on controlled-pore glass beads followed by the binding of anti-OTA antibody, with anti-IgG antibody peroxidase conjugate used as a label. In this manner, an original bio-sensing material was obtained. Upon incubation of this material with OTA, the toxin competes with OTB for the binding sites of the anti-OTA antibodies and releases the antibody-tagged peroxidase complex into the solution. Compared to classic competitive immunoassays, this newly developed displacement immunoassay presents a similar detection limit and assay time. Moreover, the detection, based on the activity of the horseradish peroxidase, is performed for the first time in situ using wine samples.

Monitoring of glucose and glutamate using enzyme microstructures and scanning electrochemical microscopy.

Glucose oxidase and glutamate oxidase lines, with typical width of 100 microm, were patterned on gold surfaces using a micro-dispensing system, by shooting 100 pl droplets of the corresponding enzyme solutions. The probe of a scanning electrochemical microscope (SECM) was then carefully positioned in the close proximity of the enzyme microstructure and poised to +600 mV vs. Ag/AgCl, KCl 0.1 M. The H(2)O(2), generated by the enzyme lines at different concentrations of glucose and glutamate in the surrounding solution, was sequentially monitored. Reproducible calibration curves for glucose and glutamate were obtained in one single experiment, proving that the combination of enzyme microstructures with SECM can provide a new way of achieving multianalyte detection.

Phenols monitoring and Hill coefficient evaluation using tyrosinase-based amperometric biosensors.

Sensitive amperometric biosensors for phenols compounds, based on tyrosinase (polyphenoloxidase, PPO) immobilized on a Pt electrode in an electropolymerized poly-amphiphilic pyrrole matrix or cross-linked with glutaraldehyde, were constructed and compared. Steady-state amperometric measurements, performed at -50 mV vs. SCE in aqueous phosphate buffer containing LiClO(4) 0.1 M (pH 7) as well as in a chloroform solution containing 0.1 M C(6)H(5)CH(2)N(CH(3))(3)Cl, were used in order to compare the electroanalytical and kinetic parameters of the investigated amperometric biosensors in aqueous and nonaqueous media. It was established that the polypyrrole matrix has a higher efficiency for enzyme retention resulting in higher bioelectrode sensitivity, both in aqueous buffer (690 microA M(-1)) and in chloroform (149 microA M(-1)).

Biosensors for phenol derivatives using biochemical signal amplification.

Two different approaches, both exploiting two enzymes cooperative functioning, to enhance the sensitivity of tyrosinase (PPO) based biosensor for amperometric detection of phenols have been compared. For this purpose, one monoenzyme electrode (PPO) and two bienzyme electrodes (PPO and d-glucose dehydrogenase, GDH; PPO and horseradish peroxidase, HRP) were constructed using agar-agar gel as enzyme immobilization matrix. The biosensors responses for l-tyrosine detection were recorded at -50 mV versus saturated calomel electrode (SCE). The highest sensitivity (74 mA M(-1)) was observed for the PPO-GDH couple, while that recorded for PPO-HRP couple system was only 32 times higher than that measured for monoenzyme electrode (0.01 mA M(-1)). The ability of the PPO-, PPO-GDH-, PPO-HRP-based biosensors to assay phenols was demonstrated by quantitative determination of phenol, 1,2-dihydroxybenzene, 1,3-dihydroxybenzene, 1,4-dihydroxybenzene, 2-amino-3 (4-hydroxyphenyl) propanoic acid, 2-hydroxytoluene, 3-hydroxytoluene, 4-hydroxytoluene, 4-clorophenol, 3-clorophenol, 2-clorophenol, 4-hydroxybenzoic acid.

Sensitive detection of organophosphorus pesticides using a needle type amperometric acetylcholinesterase-based bioelectrode. Thiocholine electrochemistry and immobilised enzyme inhibition.

An acetylcholinesterase (AChE) based amperometric bioelectrode for a selective detection of low concentrations of organophosphorus pesticides has been developed. The amperometric needle type bioelectrode consists of a bare cavity in a PTFE isolated Pt-Ir wire, where the AChE was entrapped into a photopolymerised polymer of polyvinyl alcohol bearing styrylpyridinium groups (PVA-SbQ). Cyclic voltammetry, performed at Pt and AChE/Pt disk electrodes, confirmed the irreversible, monoelectronic thiocholine oxidation process and showed that a working potential of +0.410 V vs. Ag/AgCl, KCl(sat) was suitable for a selective and sensitive amperometric detection of thiocholine. The acetylthiocholine detection under enzyme kinetic control was found in the range of 0.01-0.3 U cm(-2) of immobilised AChE. The detection limit, calculated for an inhibition ratio of 10%, was found to reach 5 microM for dipterex and 0.4 microM for paraoxon. A kinetic analysis of the AChE-pesticide interaction process using Hanes-Woolf or Lineweaver-Burk linearisations and secondary plots allowed identification of the immobilised enzyme inhibition process as a mixed one (non/uncompetitive) for both dipterex and paraoxon. The deviation from classical Michaelis Menten kinetics induced from the studied pesticides was evaluated using Hill plots.