Protein stability plays an important role in a wide variety of settings ranging from industrial processes where proteins are used as biocatalysts to medical settings where misfolded proteins are implicated in disease. Understanding protein stability will allow design of improved bioprocess and pharmaceutical formulations as well as aid in the development of therapies for protein-based diseases. The effects of dissolved salts on protein kinetic stability are studied here. We find that ion-solvent interactions, characterized by the Jones-Dole B-viscosity coefficient, are strong indicators of salt effects on protein deactivation. This finding is used to develop a model for predicting protein deactivation in salt solutions in terms of two competing processes. Since protein unfolding and aggregation can lead to a number of protein misfolding diseases, we test the applicability of our model for describing salt effects on transthyretin aggregation. As the factors contributing to protein stability become more understood, the use of enzymes as biocatalyst for industrial process will increase, and the need for enzymes active in a wide range of reaction media will increase. We have developed a process using an enzyme in combination with organic-aqueous tunable solvents (OATS) which allows for monophasic reaction of the enzyme with hypdrophobic substrates. The reaction mixture can be separated into two phases by the addition of carbon dioxide pressure. This separation allows for both convenient recovery of the hydrophobic reaction product from the organic phase as well as recycle of the enzyme in the aqueous phase. Overall reaction conversions of 80% and little enzyme activity loss are observed after six reaction cycles.

The objective of this study was to investigate the possibility of recycling enzymes during saccharification of cellulose for the production of ethanol from woodchips. To make enzyme recycling feasible and economical when woodchips are processed for ethanol, the lignin in the wood is to be removed before the enzymes are added. Since enzymes constitute a major part of the input costs, second only to the feedstock, the ability to reuse the enzymes could lead to a considerable decrease in the production cost of ethanol. Tulip poplar woodchips were selected as the feedstock. Different delignification methods with recovery of byproducts were investigated. Alkali extraction, using dilute NaOH for the removal of lignin after steam pretreatment, was used as the base case against which all other processes were compared. Recovery of furfural and methanol, produced during the pretreatment of the woodchips, for sale as byproducts was one modification to the alkali extraction process that was investigated. The conversion of xylose to furfural and the recovery of the furfural as a byproduct was the third case explored. Solvent extraction using a 50:50 ethanol-water mixture instead of extraction with NaOH was the fourth case examined. Process flow sheets were then developed to recycle the enzymes during the hydrolysis and fermentation of this prehyrolyzed and delignified wood. Two reactor setup schemes were examined for enzyme recycling. One scheme involved a single train of reactors, with the whole pretreated slurry flowing from one reactor to the next, whereas, in the other scheme, the slurry was split among parallel trains of reactors. The activity loss of the enzymes was modeled such that a part of the enzymes entering the reactor lost all their activity. The loss of activity in multiple steps, with enzymes losing only some of their activity, was also modeled. Here the enzymes entering the reactor constituted a mixture with different activities instead of all the enzymes having the same activity like in the previous single step model. Recovering methanol and furfural reduced the minimum ethanol selling price. High temperature ethanol water pretreatment and lignin extraction reduced the minimum ethanol selling price compared to the base case of steam pretreatment followed by alkali extraction. Enzyme recycling also reduces the minimum ethanol selling price. The magnitude of the price reduction depends on the recycling scheme selected and the rate of enzyme deactivation, which has not been measured.