Electrochemical Biosensors Based on Membrane-Bound Enzymes in Biomimetic Configurations.

In nature, many enzymes are attached or inserted into the cell membrane, having hydrophobic subunits or lipid chains for this purpose. Their reconstitution on electrodes maintaining their natural structural characteristics allows for optimizing their electrocatalytic properties and stability. Different biomimetic strategies have been developed for modifying electrodes surfaces to accommodate membrane-bound enzymes, including the formation of self-assembled monolayers of hydrophobic compounds, lipid bilayers, or liposomes deposition. An overview of the different strategies used for the formation of biomimetic membranes, the reconstitution of membrane enzymes on electrodes, and their applications as biosensors is presented.

Enzymatic synthesis of Hydroxytyrosol from Oleuropein for valorization of an agricultural waste.

Oleuropein (OP) is an appreciated compound present not only in fruits but also in leaves of olive trees, which can be transformed into hydroxytyrosol (HT), a substance with high antioxidant activity. In this work, the transformation of an agricultural residue containing OP (olive leaves or wastewater from mills) to the high added value compound HT is accomplished through different enzymatic strategies. Different enzymes were used, immobilized on various supports by diverse binding forces: beta-glucosidase encapsulated in siliceous material, esterases and lipases immobilized on hydrophobic supports (octyl-functionalized amorphous silica and periodic mesoporous organosilica), and esterase immobilized on amine-functionalized ordered mesoporous silica. All these biocatalysts were tested for oleuropein hydrolysis through two different reaction approaches: a) split of glucosidic bond catalyzed by beta-glucosidase (ß-glu), followed by hydrolysis of the aglycon and further ester hydrolysis. 5 mg·mL-1 of ß-glu fully hydrolyzed 5 mM OP at pH 7 and 50°C in 7 days, and further enzymatic hydrolysis of the aglycon yielded near to 0.5 mM HT in the best conditions tested. b) via direct hydrolysis of the ester bond to produce hydroxytyrosol in a one-step reaction using esterases or lipases. The latter reaction pathway catalyzed by lipase from Penicillium camemberti immobilized on octyl-silica (4 mg·mL-1) at 35°C and pH 6 directly produced 6.8 mM HT (1 mg·mL-1), transforming in 12 days near to 30% of the initial 25 mM OP from a commercial olive leaves extract.

Electro-enzymatic ATP regeneration coupled to biocatalytic phosphorylation reactions.

Adenosine-5-triphosphate (ATP) is the main energy vector in biological systems, thus its regeneration is an important issue for the application of many enzymes of interest in biocatalysis and synthetic biology. We have developed an electroenzymatic ATP regeneration system consisting in a gold electrode modified with a floating phospholipid bilayer that allows coupling the catalytic activity of two membrane-bound enzymes: NiFeSe hydrogenase from Desulfovibrio vulgaris and F1Fo-ATP synthase from Escherichia coli. Thus, H2 is used as a fuel for producing ATP. This electro-enzymatic assembly is studied as ATP regeneration system of phosphorylation reactions catalysed by kinases, such as hexokinase and NAD+-kinase for respectively producing glucose-6-phosphate and NADP+.

Potentiometric detection of ATP based on the transmembrane proton gradient generated by ATPase reconstituted on a gold electrode.

Adenosine triphosphate (ATP) is a key molecule as energy vector for living organisms, therefore its detection reveals the presence of microbial colonies. Environments where the existence of microbial pathogens suppose a health hazard can benefit from real time monitoring of such molecule. We report a potentiometric biosensor based on ATP-synthase from Escherichia coli reconstituted in a floating phospholipid bilayer over gold electrodes modified with a 4-aminothiophenol self-assembled monolayer. The use of a pH-dependent redox probe on the electrode surface allows a simple, specific and reliable on site determination of ATP concentration from 1 µM to 1 mM. The broad range ATP biosensor can offer an alternative way of measuring in a few minutes the presence of microbial contamination.

A new mechanistic model for an O2-protected electron-bifurcating hydrogenase, Hnd from Desulfovibrio fructosovorans.

The genome of the sulfate-reducing and anaerobic bacterium Desulfovibrio fructosovorans encodes different hydrogenases. Among them is Hnd, a tetrameric cytoplasmic [FeFe] hydrogenase that has previously been described as an NADP-specific enzyme (Malki et al., 1995). In this study, we purified and characterized a recombinant Strep-tagged form of Hnd and demonstrated that it is an electron-bifurcating enzyme. Flavin-based electron-bifurcation is a mechanism that couples an exergonic redox reaction to an endergonic one allowing energy conservation in anaerobic microorganisms. One of the three ferredoxins of the bacterium, that was named FdxB, was also purified and characterized. It contains a low-potential (Em = -450 mV) [4Fe4S] cluster. We found that Hnd was not able to reduce NADP+, and that it catalyzes the simultaneous reduction of FdxB and NAD+. Moreover, Hnd is the first electron-bifurcating hydrogenase that retains activity when purified aerobically due to formation of an inactive state of its catalytic site protecting against O2 damage (Hinact). Hnd is highly active with the artificial redox partner (methyl viologen) and can perform the electron-bifurcation reaction to oxidize H2 with a specific activity of 10 µmol of NADH/min/mg of enzyme. Surprisingly, the ratio between NADH and reduced FdxB varies over the reaction with a decreasing amount of FdxB reduced per NADH produced, indicating a more complex mechanism than previously described. We proposed a new mechanistic model in which the ferredoxin is recycled at the hydrogenase catalytic subunit.