Towards cell-free isobutanol production: Development of a novel immobilized enzyme system.

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.

Stability of proteins on hydrophilic surfaces.

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.

Stability of proteins inside a hydrophobic cavity.

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.