We study the effects of confinement and hydrophobicity of a spherical cavity on the structural and thermal stability of proteins in the framework of a hydrophobic-polar (HP) lattice model. We observe that a neutral confinement stabilizes the folded state of the protein by eliminating many of the open-chain conformations of the unfolded state. Hydrophobic confinement always destabilizes the protein because of protein-surface interactions. However, for moderate surface hydrophobicities, the protein remains stabilized relative to its state in free solution because of the dominance of entropic effects. These results are consistent with our experimental findings of (a) enhanced activity of alcohol dehydrogenase (ADH) when immobilized inside the essentially cylindrical pores of hydrophilic mesoporous silica (SBA-15) and (b) unaffected activity when immobilized inside weakly hydrophobic pores of methacrylate resin compared to its activity in free solution. In the same vein, our predictions are also consistent with the behavior of lysozyme and myoglobin in hydrophilic and hydrophobic SBA-15, which show qualitatively the same trends. Apparently, our results have validity across these very different enzymes, and we therefore suggest that confinement can be used to selectively improve enzyme performance.

Isolating and concentrating volatile organics such as fuels and intermediate chemicals from aqueous solutions is important for environmental synthesis and processing. We have prepared a new class of easy-to-synthesize polymeric membranes comprising hydrophobic brush-like structures as a selective layer, and have tested them using pervaporation of isobutanol from water. These brush structures were prepared by graft-induced polymerization of hydrophobic vinyl monomers from light-sensitive poly(ether sulfone) nanofiltration support membranes (grafting from) without initiating agents. Graft-induced tethered polymer chains with multiple C18 alkane side-chains out-performed the industry gold standard silicone rubber membrane with selectivities of α = 10.1 ± 0.9 and 6.7 ± 0.1, respectively, at comparable permeation fluxes of 0.7− 1.0 ± 0.1 L/m 2 -h. Preparation of these brush membranes is simpler and easier to scale-up than current methods of preparing asymmetric and composite membrane structures. These brush structures and this method of preparation have excellent potential for synthesizing selective membranes suitable for large-scale organic−water separations. ■ INTRODUCTIONPressure-driven membrane filtration processes have matured and are now widely accepted in many industries such as in energy, biotechnology, food and beverage, chemical, wastewater, gas fractionation, and desalination due to their low energy requirements and one-phase operation. Although membranes made from metals or ceramics are available, polymeric materials predominate. 1 For over 40 years, both interfacial polymerization 2 and phase inversion 3 have been the predominant methods for preparing composite polymeric and asymmetric membrane structures, respectively. Although these synthesis methods have been very successful, they are both relatively complex and sensitive to small changes in the casting conditions. 4 Many research groups have sought novel synthesis methods for producing synthetic membranes without much success. These membranes suffer from limitations including low porosity (track etched), 5−7 high cost (ceramic or stainless steel), 8 wide pore size distribution (stretched PTFE), 9−11 and low strength (biological), 12 or are difficult to scale-up (zeolite, carbon-nanotubes or graphene oxide). 13−15 Here, we synthesize a new class of synthetic brush hydrophobic polymer membranes, and then test them with a challenging separation: removal of isobutanol from water by pervaporation (PV). 16 To prepare the best performing hydrophobic brush membranes for this separation, we used our unique high throughput platform with 96 filter well plates. 17 The method of preparation involves grafting commercially available vinyl monomers alone or in mixtures to light-sensitive poly(ether sulfone) (PES) nanofiltration membranes, screening for the best performers, and selecting the winners. 18−20 The high throughput platform has also been used with combinatorial chemistry to generate a library of new monomers. 19,21 The newly synthesized hydrophobic-terminat...

The physical and chemical properties of solid substrates or surfaces critically influence the stability and activity of immobilized proteins such as enzymes. Reports of increased stability and activity of enzymes near/on surfaces as compared with those in solution abound; however, a mechanistic understanding is wanting. Simulations and experiments are used here to provide details toward such a mechanistic understanding. Experiments demonstrate increased activity of alcohol dehydrogenase (ADH) inside moderate hydrophilic mesopourous silica (SBA-15) pores but drastically decreased activity inside very hydrophilic NH2-SBA-15 surfaces as compared with that in solution. Also, the temperature stability of ADH was increased over that in solution when immobilized in a cavity with a mildly hydrophilic surface. Simulations confirm these experimental findings. Simulations calculated in the framework of a hydrophobic-polar (H-P) lattice model show increased thermal stability of a model 64-mer peptide on positive and zero curvature surfaces over that in solution. Peptides immobilized inside negative curvature cavities (concave) with hydrophilic surfaces exhibit increased stability only inside pores that are only 3-4 nm larger than the hydrodynamic radius of the peptide. Peptides are destabilized, however, when the surface hydrophilic character inside very small cavities/pores becomes large.

To overcome the main challenges facing alcohol-based biofuel production, we propose an alternate simplified biofuel production scheme based on a cell-free immobilized enzyme system. In this paper, we measured the activity of two tetrameric enzymes, a control enzyme with a colorimetric assay, β-galactosidase, and an alcohol-producing enzyme, alcohol dehydrogenase, immobilized on multiple surface curvatures and chemistries. Several solid supports including silica nanoparticles (convex), mesopourous silica (concave), diatomaceous earth (concave), and methacrylate (concave) were examined. High conversion rates and low protein leaching was achieved by covalent immobilization of both enzymes on methacrylate resin. Alcohol dehydrogenase (ADH) exhibited long-term stability and over 80% conversion of aldehyde to alcohol over 16 days of batch cycles. The complete reaction scheme for the conversion of acid to aldehyde to alcohol was demonstrated in vitro by immobilizing ADH with keto-acid decarboxylase free in solution.

Producing fuels and chemical intermediates with cell cultures is severely limited by low product concentrations (≤0.2%(v/v)) due to feedback inhibition, cell instability, and lack of economical product recovery processes. We have developed an alternate simplified production scheme based on a cell-free immobilized enzyme system. Two immobilized enzymes (keto-acid decarboxylase (KdcA) and alcohol dehydrogenase (ADH)) and one enzyme in solution (formate dehydrogenase (FDH) for NADH recycle) produced isobutanol titers 8 to 20 times higher than the highest reported titers with S. cerevisiae on a mol/mol basis. These high conversion rates and low protein leaching were achieved by covalent immobilization of enzymes (ADH) and enzyme fusions (fKdcA) on methacrylate resin. The new enzyme system without in situ removal of isobutanol achieved a 55% conversion of ketoisovaleric acid to isobutanol at a concentration of 0.135 (mole isobutanol produced for each mole ketoisovaleric acid consumed). Further increasing titer will require continuous removal of the isobutanol using an in situ recovery system.