Marcos Pita

Biocomputing Security System: Concatenated Enzyme-Based Logic Gates Operating as a Biomolecular Keypad Lock

A biomolecular security system mimicking a keypad lock device was developed using enzyme-based concatenated AND logic gates resulting in the implication logic network.

Novel Polyphenol Oxidase Mined from a Metagenome Expression Library of Bovine Rumen BIOCHEMICAL PROPERTIES, STRUCTURAL ANALYSIS, AND PHYLOGENETIC RELATIONSHIPS

RL5, a gene coding for a novel polyphenol oxidase, was identified through activity screening of a metagenome expression library from bovine rumen microflora. Characterization of the recombinant protein produced in Escherichia coli revealed a multipotent capacity to oxidize a wide range of substrates (syringaldazine > 2,6-dimethoxyphenol > veratryl alcohol > guaiacol > tetramethylbenzidine > 4-methoxybenzyl alcohol > 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) >> phenol red) over an unusually broad range of pH from 3.5 to 9.0. Apparent Km and kcat values for ABTS, syringaldazine, and 2,6-dimetoxyphenol obtained from steady-state kinetic measurements performed at 40 °C, pH 4.5, yielded values of 26, 0.43, and 0.45 μm and 18, 660, and 1175 s-1, respectively. The Km values for syringaldazine and 2,6-dimetoxyphenol are up to 5 times lower, and the kcat values up to 40 times higher, than values previously reported for this class of enzyme. RL5 is a 4-copper oxidase with oxidation potential values of 745, 400, and 500 mV versus normal hydrogen electrode for the T1, T2, and T3 copper sites. A three-dimensional model of RL5 and site-directed mutants were generated to identify the copper ligands. Bioinformatic analysis of the gene sequence and the sequences and contexts of neighboring genes suggested a tentative phylogenetic assignment to the genus Bacteroides. Kinetic, electrochemical, and EPR analyses provide unequivocal evidence that the hypothetical proteins from Bacteroides thetaiotaomicron and from E. coli, which are closely related to the deduced protein encoded by the RL5 gene, are also multicopper proteins with polyphenol oxidase activity. The present study shows that these three newly characterized enzymes form a new family of functional multicopper oxidases with laccase activity related to conserved hypothetical proteins harboring the domain of unknown function DUF152 and suggests that some other of these proteins may also be laccases.

Biofuel Cell Controlled by Enzyme Logic Systems

An enzyme-based biofuel cell with a pH-switchable oxygen electrode, controlled by enzyme logic operations processing in situ biochemical input signals, has been developed. Two Boolean logic gates (AND/OR) were assembled from enzyme systems to process biochemical signals and to convert them logically into pH-changes of the solution. The cathode used in the biofuel cell was modified with a polymer-brush functionalized with Os-complex redox species operating as relay units to mediate electron transport between the conductive support and soluble laccase biocatalyzing oxygen reduction. The electrochemical activity of the modified electrode was switchable by alteration of the solution pH value. The electrode was electrochemically mute at pH > 5.5, and it was activated for the bioelectrocatalytic oxygen reduction at pH < 4.5. The sharp transition between the inactive and active states was used to control the electrode activity by external enzymatic systems operating as logic switches in the system. The enzyme logic systems were decreasing the pH value upon appropriate combinations of the biochemical signals corresponding to the AND/OR Boolean logic. Then the pH-switchable electrode was activated for the oxygen reduction, and the entire biofuel cell was switched ON. The biofuel cell was also switched OFF by another biochemical signal which resets the pH value to the original neutral value. The present biofuel cell is the first prototype of a future implantable biofuel cell controlled by complex biochemical reactions to deliver power on-demand responding in a logical way to the physiological needs.

Laccase electrode for direct electrocatalytic reduction of O2 to H2O with high-operational stability and resistance to chloride inhibition

Laccase from Trametes hirsuta basidiomycete has been covalently bound to graphite electrodes electrochemically modified with phenyl derivatives as a way to attach the enzyme molecules with an adequate orientation for direct electron transfer (DET). Current densities up to 0.5mA/cm(2) of electrocatalytic reduction of O(2) to H(2)O were obtained in absence of redox mediators, suggesting preferential orientation of the T1 Cu centre of the laccase towards the electrode. The covalent attachment of the laccase molecules to the functionalized electrodes permitted remarkable operational stability. Moreover, O(2) bioelectroreduction based on DET between the laccase and the electrode was not inhibited by chloride ions, whereas mediated bioelectrocatalysis was. In contrast, fluoride ions inhibited both direct and mediated electron transfers-based bioelectrocatalytic reduction of O(2). Thus, two different modes of laccase inhibition by halides are discussed.

Novel Polyphenol Oxidase Mined from a Metagenome Expression Library of Bovine Rumen

RL5, a gene coding for a novel polyphenol oxidase, was identified through activity screening of a metagenome expression library from bovine rumen microflora. Characterization of the recombinant protein produced in Escherichia coli revealed a multipotent capacity to oxidize a wide range of substrates (syringaldazine > 2,6-dimethoxyphenol > veratryl alcohol > guaiacol > tetramethylbenzidine > 4-methoxybenzyl alcohol > 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) >> phenol red) over an unusually broad range of pH from 3.5 to 9.0. Apparent Km and kcat values for ABTS, syringaldazine, and 2,6-dimetoxyphenol obtained from steady-state kinetic measurements performed at 40 °C, pH 4.5, yielded values of 26, 0.43, and 0.45 μm and 18, 660, and 1175 s-1, respectively. The Km values for syringaldazine and 2,6-dimetoxyphenol are up to 5 times lower, and the kcat values up to 40 times higher, than values previously reported for this class of enzyme. RL5 is a 4-copper oxidase with oxidation potential values of 745, 400, and 500 mV versus normal hydrogen electrode for the T1, T2, and T3 copper sites. A three-dimensional model of RL5 and site-directed mutants were generated to identify the copper ligands. Bioinformatic analysis of the gene sequence and the sequences and contexts of neighboring genes suggested a tentative phylogenetic assignment to the genus Bacteroides. Kinetic, electrochemical, and EPR analyses provide unequivocal evidence that the hypothetical proteins from Bacteroides thetaiotaomicron and from E. coli, which are closely related to the deduced protein encoded by the RL5 gene, are also multicopper proteins with polyphenol oxidase activity. The present study shows that these three newly characterized enzymes form a new family of functional multicopper oxidases with laccase activity related to conserved hypothetical proteins harboring the domain of unknown function DUF152 and suggests that some other of these proteins may also be laccases.

Gold Nanoparticles as Electronic Bridges for Laccase-Based Biocathodes

Direct electron transfer (DET) reactions between redox enzymes and electrodes can be maximized by oriented immobilization of the enzyme molecules onto an electroactive surface modified with functionalized gold nanoparticles (AuNPs). Here, we present such strategy for obtaining a DET-based laccase (Lc) cathode for O(2) electroreduction at low overpotentials. The stable nanostructured enzymatic electrode is based on the step-by-step covalent attachment of AuNPs and Lc molecules to porous graphite electrodes using the diazonium salt reduction strategy. Oriented immobilization of the enzyme molecules on adequately functionalized AuNPs allows establishing very fast DET with the electrode via their Cu T1 site. The measured electrocatalytic waves of O(2) reduction can be deconvoluted into two contributions. The one at lower overpotentials corresponds to immobilized Lc molecules that are efficiently wired by the AuNPs with a heterogeneous electron transfer rate constant k(0) ≫ 400 s(-1).

Synthesis of cobalt ferrite core/metallic shell nanoparticles for the development of a specific PNA/DNA biosensor

Controlled synthesis of cobalt ferrite superparamagnetic nanoparticles covered with a gold shell has been achieved by an affinity and trap strategy. Magnetic nanoparticles are functionalized with a mixture of amino and thiol groups that facilitate the electrostatic attraction and further chemisorption of gold nanoparticles, respectively. Using these nanoparticles as seeds, a complete coating shell is achieved by gold salt-iterative reduction leading to monodisperse water-soluble gold-covered magnetic nanoparticles, with an average diameter ranging from 21 to 29 nm. These constitute a versatile platform for immobilization of biomolecules via thiol chemistry, which is exemplified by the immobilization of peptide nucleic acid (PNA) oligomers that specifically hybridize with complementary DNA molecules in solution. Hybridation with DNA probes has been measured using Rhodamine 6G fluorescence marker and the detection of a single nucleotide mutation has been achieved. These results suggest the PNA-nanoparticles application as a biosensor for DNA genotyping avoiding commonly time-consuming procedures employed.

Biochemically controlled bioelectrocatalytic interface.

A switchable bioelectrocatalytic system for glucose oxidation controlled by external biochemical signals exemplifies interfacing between bioelectronic and biochemical ensembles.

Boolean logic gates that use enzymes as input signals.

Biochemical systems that demonstrate the Boolean logic operations AND, OR, XOR, and InhibA were developed by using soluble compounds, which represent the chemical “devices”, and the enzymes glucose oxidase (GOx), glucose dehydrogenase (GDH), alcohol dehydrogenase (AlcDH), and microperoxidase‐11 (MP‐11), which operated as the input signals that activated the logic gates. The enzymes were used as soluble materials and as immobilized biocatalysts. The studied systems are proposed to be a step towards the construction of “smart” signal‐responsive materials with built‐in Boolean logic.

Self-powered wireless carbohydrate/oxygen sensitive biodevice based on radio signal transmission

Here for the first time, we detail self-contained (wireless and self-powered) biodevices with wireless signal transmission. Specifically, we demonstrate the operation of self-sustained carbohydrate and oxygen sensitive biodevices, consisting of a wireless electronic unit, radio transmitter and separate sensing bioelectrodes, supplied with electrical energy from a combined multi-enzyme fuel cell generating sufficient current at required voltage to power the electronics. A carbohydrate/oxygen enzymatic fuel cell was assembled by comparing the performance of a range of different bioelectrodes followed by selection of the most suitable, stable combination. Carbohydrates (viz. lactose for the demonstration) and oxygen were also chosen as bioanalytes, being important biomarkers, to demonstrate the operation of the self-contained biosensing device, employing enzyme-modified bioelectrodes to enable the actual sensing. A wireless electronic unit, consisting of a micropotentiostat, an energy harvesting module (voltage amplifier together with a capacitor), and a radio microchip, were designed to enable the biofuel cell to be used as a power supply for managing the sensing devices and for wireless data transmission. The electronic system used required current and voltages greater than 44 µA and 0.57 V, respectively to operate; which the biofuel cell was capable of providing, when placed in a carbohydrate and oxygen containing buffer. In addition, a USB based receiver and computer software were employed for proof-of concept tests of the developed biodevices. Operation of bench-top prototypes was demonstrated in buffers containing different concentrations of the analytes, showcasing that the variation in response of both carbohydrate and oxygen biosensors could be monitored wirelessly in real-time as analyte concentrations in buffers were changed, using only an enzymatic fuel cell as a power supply.