Arnon Heyman

Enhanced cellulose degradation by nano-complexed enzymes: Synergism between a scaffold-linked exoglucanase and a free endoglucanase

Protein molecular scaffolds are attracting interest as natural candidates for the presentation of enzymes and acceleration of catalytic reactions. We have previously reported evidence that the stable protein 1 (SP1) from Populustremula can be employed as a molecular scaffold for the presentation of either catalytic or structural binding (cellulosomal cohesin) modules. In the present work, we have displayed a potent exoglucanase (Cel6B) from the aerobic cellulolytic bacterium, Thermobifida fusca, on a cohesin-bearing SP1 scaffold. For this purpose, a chimaeric form of the enzyme, fused to a cellulosomal dockerin module, was prepared. Full incorporation of 12 dockerin-bearing exoglucanase molecules onto the cohesin-bearing scaffold was achieved. Cellulase activity was tested on two cellulosic substrates with different levels of crystallinity, and the activity of the scaffold-linked exoglucanase was significantly reduced, compared to the free dockerin-containing enzyme. However, addition of relatively low concentrations of a free wild-type endoglucanase (T. fusca Cel5A) that bears a cellulose-binding module, in combination with the complexed exoglucanase resulted in a marked rise in activity on both cellulosic substrates. The endoglucanase cleaves internal sites of the cellulose chains, and the new chain ends of the substrate were now readily accessible to the scaffold-borne exoglucanase, thereby resulting in highly effective, synergistic degradation of cellulosic substrates.

Improved Adhesives Containing CNT/SP1 Nano Fillers

Carbon nanotubes (CNT) have stimulated research due to their wide range of applications. However, their existence as aggregates and the difficulty in debundling and dispersion limits the improvement of properties when used as fillers. Many techniques have been employed to obtain such dispersions including mechanical, ultrasonic, and solution mixing, resulting in limited effect. Attaching a protein moiety such as SP1 showed promising results. SP1 is a thermally stable protein, originally isolated from poplar trees, which self-assembles to an extremely stable 11-nm ring-shape dodecamer. Linkage of CNT to specific peptides on SP1 N-terminus by genetic engineering resulted in 12 CNT binding sites per ring. It was demonstrated by us that the CNT/SP1 complex prevents CNT aggregation and allows its homogenous mixing in water at rather low CNT/protein weight ratio (20:1). In order to obtain homogenous CNTs in a polymer matrix, the dehydrated complex was redispersed in epoxy resin. The CNT/SP1 is covalently bound to epoxy groups prior to polymerization with the curing agent. Dispersion and uniformity were improved by using a speed-mixer and a 3-roll mill. CNT/SP1 in epoxy resin exhibited improved mechanical properties compared with pure unfilled epoxy (EPON® 828/Versamide® 140). CNT/SP1 filler in epoxy adhesive at less than 1% wt. improved peel strength by 50% and shear strength by 24%. In addition, HR-SEM images of 0.7% wt. CNT/SP1 nano-filled epoxy adhesive fracture surfaces demonstrates efficient load transfer and crack arrest by the CNT/SP1 particles. Moreover, comparing the thermal properties of neat epoxy with those of 0.35% and 0.7% wt. CNT/SP1 filled nano-composite was tested using three methods: differential scanning calorimetry (DSC), thermomechanical analysis (TMA), and thermogravimetric analysis (TGA) showed a dramatic improvement increasing Tg by 20°C.

Logic implementations using a single nanoparticle–protein hybrid

A Set-Reset machine is the simplest logic circuit with a built-in memory. Its output is a (nonlinear) function of the input and of the state stored in the machines memory. Here, we report a nanoscale Set-Reset machine operating at room temperature that is based on a 5-nm silicon nanoparticle attached to the inner pore of a stable circular protein. The nanoparticle-protein hybrid can also function as a balanced ternary multiplier. Conductive atomic force microscopy is used to implement the logic input and output operations, and the processing of the logic Set and Reset operations relies on the finite capacitance of the nanoparticle provided by the good electrical isolation given by the protein, thus enabling stability of the logic device states. We show that the machine can be cycled, such that in every successive cycle, the previous state in the memory is retained as the present state. The energy cost of one cycle of computation is minimized to the cost of charging this state.

Wiring of Redox Enzymes on Three Dimensional Self-Assembled Molecular Scaffold

The integration of biological molecules and nanoscale components provides a fertile basis for the construction of hybrid materials of synergic properties and functions. Stable protein 1 (SP1), a highly stable ring shaped protein, was recently used to display different functional domains, to bind nanoparticles (NPs), and to spontaneously form two and three-dimensional structures. Here we show an approach to wire redox enzymes on this self-assembled protein-nanoparticle hybrid. Those hybrids are genetically engineered SP1s, displaying glucose oxidase (GOx) enzymes tethered to the protein inner pore. Moreover, the Au-NP-protein hybrids self-assembled to multiple enzymatic layers on the surface. By wiring the redox enzymes to the electrode, we present an active structure for the bioelectrocatalytic oxidation of glucose. This system demonstrates for the first time a three-dimensional assembly of multiple catalytic modules on a protein scaffold with an efficient electrical wiring of the enzyme units on an electrode surface, thus implementing a hybrid electrically active unit for nanobioelectronic applications.

Formation of Hydrophilic Nanochannels in the Membrane of Living Cells by the Ringlike Stable Protein-SP1

The assembly of functional junction between nerve cells and electronic sensing pads is a critical problem in the construction of effective neuroelectronic hybrid systems. Here, we demonstrate for the first time that the ringlike Stable Protein 1 (Sp1) and its derivatives can be used to generate hydrophilic nanochannels in the plasma membrane of living cells. Since SP1-derivatives can be linked to both the plasma membrane, gold or silicon surfaces, they may serve to ohmically link between cells interior and electronic sensing devices.

Float and Compress : Honeycomb-like Array of a Highly Stable Protein Scaffold

Organizing nano-objects, proteins in particular, on surfaces is one of the primary goals of bio/chemical nanotechnology. A highly stable protein scaffold (6His-SP1) was organized into a hexagonal 2D array by a new, versatile method. The protein was expelled from solution into the air/water interface and compressed in a Langmuir trough into a closely packed monolayer without the use of phospholipids or other surfactants at the interface. The 2D arrays formed at the air/water interface were characterized by transmission electron microscopy (TEM) and atomic force microscopy (AFM).