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Electrosomes preparation and application

The document describes electrosomes, which are a novel surface display system composed of enzymes attached to a scaffoldin protein. This allows for multiple electron release from fuel oxidation. In the anode, an ethanol oxidation cascade is assembled using alcohol dehydrogenase and formaldehyde dehydrogenase enzymes attached to the scaffoldin. In the cathode, copper oxidase is attached for oxygen reduction. The electrosomes provide advantages as a fuel cell and drug delivery system by catalyzing chemical energy conversion to electricity and providing controlled drug release.

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Preparation And Application Of Electrosomes GUIDED BY: Dr. Beny Baby HOD And Assistant Professor Department of Pharmaceutics Presented by: Mahesh Kumar Mishra M. Pharm 2nd Semester Department of Pharmaceutics
Contents  Introduction  Objectives of Electrosomes  Method of preparation  Advantages Electrosomes  Disadvantages Electrosomes  Application  Reference
Introduction
 The electrosomes, a novel surface-display system based on the specific interaction between the cellulosomal scaffoldin protein and a cascade of redox enzymes that allows multiple electron-release by fuel oxidation. The electrosomes is composed of two compartment:(i) a hybrid anode, which consists of dockerin-containing enzymes attached specifically to cohesin sites in the scaffoldin to assemble an ethanol oxidation cascade, and (ii) a hybrid cathode, which consists of a dockerin-containing oxygen-reducing enzyme attached in multiple copies to the cohesin-bearing scaffoldin.  The electrosomes was designed for use both in an anode and a cathode compartment; in each compartment, the unique attributes of the cellulosome scaffoldin give a different advantage.
 In the anode, the ethanol oxidation cascade consists of two enzymes, ADH and formaldehyde dehydrogenase (FormDH), both containing a different dockerin module of Acetivibrio cellulolyticus and of Clostridium thermocellum, C. thermocellum (zADH-Ac and pFormDH-Ct), respectively, assembled on a ‘designer’-scaffoldin chimera displayed on the surface of S. cerevisiae.  At the cathode, copper oxidase (CueO) was selected for surface-display. CueO is a multi-copper oxidase enzyme expressed by E. coli that catalyzes the oxidation of Cu(I) ions coupled to oxygen reduction to water.  The different constructs used for assembly are depicted. We report the characterization of the dockerin-containing enzymes and their electrochemical activity using a diffusing redox mediator.
Objective of Electrosomes  The molecular roots of our actions and the thoughts and feelings that drive us to act are ion channels, proteins that form macromolecular pores in cell membranes. These transmembrane proteins generate and propagate the electrical signals that allow us to sense our surroundings, process information, make decisions, and move.  Ion channel proteins act as gates that span the lipid bilayer that surrounds all cells where they open and close to allow the flow of ions down their electrochemical gradients.  The ion flux through a channel pore can be extremely high, ≈106 ions per second. Because of their central role in the function of the excitable tissues such as heart, brain, muscles, and nervous system, investigators have long sought to understand ion channel properties from a molecular perspective.
 The pore-forming domains of most ion channels are multimeric assemblies possessing cyclic symmetry in a general architecture known as barrel-stave. A fixed number of subunits assemble around the axis of the ion conduction pore.  The very nature of these molecules—transmembrane proteins that are difficult to obtain in the large quantities and high purity necessary for structural investigation—has impeded attempts to obtain the most essential information for understanding their functions, a three-dimensional description of their molecular architectures at high resolution.
Method of Preparation 1. Strains and Constructs method. 2. Enzyme Binding to Scaffoldin. 3. Biofuel-Cell Assembly and Characterization. 4. Protein Expression. 5. Enzyme Activity Assays. 6. Construction of YSD of Chimeric Scaffoldins. 7. Cyclic Voltammetry (CV) and Chronoamperometry (CA).
Strains and Constructs method  The genes encoding dockerins of Acetivibrio cellulolyticus and Clostridium thermocellum were cloned and ligated to the C-terminus of Zymomonas mobilis alcohol dehydrogenase and to Pseudomonasputida formaldehyde dehydrogenase by standard methods.  The dockerin module of C.thermocellum was also ligated to the C-terminus of CueO (CueO-Ct) of E.coli.  All the dockerin-containing enzymes encoding genes have been cloned into the pET15b vector for expression in E. coli, yielding the pET15b-zADH-Ac, pET15b- pFormDH-Ct, and pET15b-CueO-Ct vectors.  For controls, the genes encoding the native enzymes without an appended dockerin module were also cloned in the same vector, yielding plasmids pET15b- zADH, pET15b-pFormDH, and pET15b-CueO.  All the chemicals used in this study are detailed in the SI section.
Enzyme Binding to Scaffoldin  2.0 mL of yeast cells displaying scaffoldin, for which absorbance at a wavelength of 600 nm was 1.0 , were incubated with bacterial lysates containing the expressed enzymes at room temperature for 1 h. 1.0 mL of the bacterial lysates were used for the binding, which was performed in a final volume of 15 ml.  As a binding buffer, 50 mM Tris buffer at pH 8.0 with 1 mM CaCl2 was used. Upon binding, the yeast cells were precipitated, and binding was repeated using fresh lysate.  After the second binding cycle, the yeast cells were washed four times in the buffer to remove non-specifically bound enzymes.  For the CueO-Ct binding, the yeast cells were suspended in 0.1m acetate buffer pH 5.0 containing 1 mm CaCl2 after the last wash.  Following binding, the yeast cells were resuspended in 2.0 ml of buffer.
Biofuel-Cell Assembly and Characterization  Air was continuously purged to the fuel-cells. A potentiostatically controlled anode set to −0.2 V versus Ag/AgCl was used.  In all experiments, the cells were left to stabilize overnight, following fuel cell assembly, before characterization was performed.  The characterization of fuel cell performance was done by measuring the voltage of the cells under variable external loads.  A background current cell was used as a negative control for all fuel cell experiments and did not contain any yeast. Graphite rods of 5 mm diameter served as both anodes and cathodes.  The counter electrode that served for the potentiostatically controlled electrode was of a larger surface area, as described for the CV and CA measurements.
Advantages  It perpetuates the endurance of active drug molecule in the systemic circulation.  Deferment the elimination reactions of promptly metabolize drugs and contributes to controlled release.  Incorporates both hydrophilic and lipophilic drugs.  Intensifies the stability of medicament.  Cost of therapy is minimized by reducing the dose per unit formulation  Elevate bioavailability especially in water disfavouring drugs.  Selective uptake by tissues due to direct drug delivery.
Disadvantages  The production cost of electrosomes are generally high since these come under the class of nanotherapeutics.  The constituent phospholipids present in lipid vesicular structures may undergo oxidation or hydrolysis.
Application  They use enzymatic reactions to catalyze the conversion of chemical energy to electricity in a fuel cell.  The use of enzymatic cascades in enzymatic fuel cell anodes resulted in very high power outputs, as the electron density achieved was much higher when the fuel was fully oxidized.  Its used as a carrier in drug targeting.  Used in the treatment of cancer.  Used in studying immune response.  Ear targeting  Muscle targeting
Reference 1. SHEFRIN S, SREELAXMI C. S, VISHNU VIJAYAN, SREEJA C. NAIR.ENZYMOSOMES: A RISING EFFECTUAL TOOL FOR TARGETED DRUG DELIVERY SYSTEM.Int J App Pharm. 2017;9(6);1-9 2. Szczupak A, Aizik D, Moraïs S, Vazana Y, Barak Y, Bayer A E, Alfonta L. The Electrosome: A Surface-Displayed Enzymatic Cascade in a Biofuel Cell’s Anode and a High-Density Surface-Displayed Biocathodic Enzyme.Nano- material.2017 3. www.science direct.com
Electrosomes preparation and application