Lidija Tetianec

Specificity and kinetic parameters of recombinant Microdochium nivale carbohydrate oxidase

Abstract A new carbohydrate oxidase from Microdochium nivale heterologously expressed in Aspergillus oryzae (rMnO) has been characterized. The carbohydrate oxidase is a flavoenzyme which oxidizes glucose and other mono- or oligosaccharides. It shows a broad substrate specificity towards carbohydrates reacting with aldoses in the 1-position. The rMnO oxidizes the β-form of d -glucose, and the product of d -glucose oxidation is d -gluconic acid. The mechanism of carbohydrate oxidation by oxygen and artificial electron acceptors has been described by a ping-pong scheme. Compared to Aspergillus niger glucose oxidase (GOx) the reactivity of rMnO at pH 7.0 is significantly lower; k cat is 20, k ox 11 and k red 22 times less, using oxygen as electron acceptor. Also with other two electron acceptors, like DPIP, the activity is low. However, compared to oxygen the rMnO shows 2–10 times higher activity towards some artificial single electron acceptors (AAs). The enzyme activity increases at higher ionic strength of the solution, if positively-charged AAs are used. The high activity towards AAs and low rate for oxygen as well as broad specificity to carbohydrates indicates that rMnO may have some advantages compared to the most used GOx in connection with enzyme use for analytical devices and for biotechnological purposes.

Pyrroloquinoline quinone-dependent carbohydrate dehydrogenase: activity enhancement and the role of artificial electron acceptors.

Pyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenase (PQQ-GDH) offers a variety of opportunities for applications, e.g. in highly sensitive biosensors and electrosynthetic reactions. Here we report on the acceleration (up to 4.9 x 10(4)-fold) of enzymatic ferricyanide reduction by artificial redox mediators (enhancers). The reaction mechanism includes reduction of the PQQ-GDH by glucose followed by oxidation of the reduced PQQ cofactor with either ferricyanide or a redox mediator. A synergistic effect occurs through the oxidation of a reduced mediator by ferricyanide. Using kinetic description of the coupled reaction, the second order rate constant for the reaction of an oxidized mediator with the reduced enzyme cofactor (k(ox)) can be calculated. For different mediators this value is 2.2 x 10(6)-1.6 x 10(8) M(-1)s(-1) at pH 7.2 and 25 degrees C. However, no correlation of the rate constant with the midpoint redox potential of the mediator could be established. For low-potential mediators the synergistic effect is proportional to the ratio of k(ox(med))/k(ox(ferricyanide)), whereas for the high-potential mediators the effect depends on both this ratio and the concentration of the oxidized mediator, which can be calculated from the Nernst equation. The described effect can be applied in various ways, e.g. for substrate reactivity determination, electrosynthetic PQQ cofactor regeneration or building of new highly sensitive biosensors.

Kinetics of N-substituted phenothiazines and N-substituted phenoxazines oxidation catalyzed by fungal laccases

Laccase-catalyzed oxidation of N-substituted phenothiazines and N-substituted phenoxazines was investigated at pH 5.5 and 25°C. The recombinant laccase from Polyporus pinsitus (rPpL) and the laccase from Myceliophthora thermophila (rMtL) were used. The dependence of initial reaction rate on substrate concentration was analyzed by applying the laccase action scheme in which the laccase native intermediate (NI) reacts with a substrate forming reduced enzyme. The reduced laccase produces peroxide intermediate (PI) which in turn decays to the NI. The calculated constant (kox) values of the PI formation are (6.1±3.1)×105 M−1s−1 for rPpL and (2.5±0.9)×104 M−1s−1 for rMtL. The bimolecular constants of the reaction of the native intermediate with electron donor (kred) vary in the interval from 2.2×105 to 2.1×107 M−1s−1 for rPpL and from 1.3×102 to 1.8×105 M-1s−1 for rMtL. The larger reactivity of rPpL in comparison to rMtL is associated with the higher redox potential of type I Cu of rPpL. The variation of kred values for both laccases correlates with the change of the redox potential of substrates. Following outer sphere (Marcus) electron transfer mechanism the calculated activationless electron transfer rate and the apparent reorganization energy are 5.0×107 M−1s−1 and 0.29 eV, respectively.

Study of the reactivity of quinohemoprotein alcohol dehydrogenase with heterocycle-pentacyanoferrate(III) complexes and the electron transfer path calculations

The reactivity of alcohol dehydrogenase IIG (ADH IIG) from Pseudomonas putida HK5 with new heterocycle-pentacyanoferrate(III) complexes and hexacyanoferrate(III) was determined at pH 7.2. The pentacyanoferrate(III) complexes contained imidazole, pyrazole, pyridine, their derivatives and 2-aminobenzothiazole as the sixth ligand. The largest reactivity of the complexes with ADH IIG was estimated for the complex containing pyridine. An apparent bimolecular constant (kox) for this complex was 8.7 × 105 M−1s−1. The lowest value of kox was estimated for the complex with benzotriazole (kox = 3.1 × 104 M−1s−1). The investigation of the hexacyanoferrate(III) enzymatic reduction rate at different ionic strength gave a single negative charge of reduced ADH IIG. Docking calculations revealed two binding sites of the complexes in ADH-IIG structure. The first one is located at the entrance to the PQQ pocket, and the second is at the site of cytochrome domain. The calculations of electron transfer (ET) path indicated that the most effective ET takes place from heme to the complex docked at the entrance to the PQQ pocket. This shortest path is constructed of amino acids Ser607 and Cys606.

The development of an improved glucose biosensor using recombinant carbohydrate oxidase from Microdochium nivale

Biosensors containing recombinant carbohydrate oxidase from Microdochium nivale (rMnO) were developed using either a chemically modified carbon paste or a graphite electrode. 1-(N,N-dimethylamine)-4-(4-morpholine)benzene (AMB) and 1,1′-dimethylferrocene (DMFc) were used as the mediators. The biosensors showed a linear calibration graph up to 0.018 mol dm−3 of glucose when operated at 0.04–0.36 V vs. SCE. Almost no change was detected in the sensitivity of the biosensors at pH 7.2–8.1. The biosensors responded to a range of D-aldoses, but maximal sensitivity of the biosensor was with D-glucose. The biosensors gave no response to polyhydroxylic compounds such as D-mannitol, D-sorbitol and inositol. The advantage of the biosensor in comparison to the biosensor based on Aspergillus niger glucose oxidase is a wide linear range, low sensitivity to oxygen and (in some cases) broad specificity.

Bioanode with alcohol dehydrogenase undergoing a direct electron transfer on functionalized gold nanoparticles for an application in biofuel cells for glycerol conversion

In this paper we designed and investigated bioanode with alcohol dehydrogenase (ADH) catalysing oxidation of glycerol and glyceraldehyde. The most effective bioanode was fabricated when ADH was immobilized on gold nanoparticles (AuNPs) modified with 4-aminothiophenol. This electrode catalysed the oxidation of both glycerol and glyceraldehyde thus demonstrating a consecutive two-step process. The bioanode generated the current density of 510µAcm-2 at pH 7.0 and 0V vs. SCE. It was demonstrated that the electrode acted effectively due to the direct electron exchange between heme of ADH and modified AuNPs. The reversible oxidation and reduction of ADH heme proceeded at around -0.05V vs. SCE. The turnover number of the immobilized enzyme was estimated to be 65s-1 which is the same as the catalytic number of the enzyme in solution. To the best of our knowledge those parameters are the highest currently reported for the alcohol dehydrogenase bioanodes operating utilizing a direct electron transfer. As a proof of biofuels cell conception, the bioanode was combined with AuNPs-laccase biocathode. The biofuel cell generated maximum power output of 130µWcm-2 at 0.5V and pH 7.0.