47244-G4
Crosslinked Enzyme Microspheres Prepared Using Liquid-Liquid Phase Separation Can Catalyze Reactions Involving Petroleum Derivatives
Cross-linked enzyme aggregates prepared using liquid-liquid phase separation can catalyze reactions involving petroleum derivatives.
In this first year, our work has focused on understanding the role of precipitating agents on enzyme precipitation and on the morphology of the obtained aggregates. This work has involved three graduate students and one undergraduate student.
We experimentally demonstrate that liquid-liquid phase separation (LLPS) of protein aqueous solutions can be induced by isothermal protein oligomerization. This phenomenon is analogous to LLPS induced by the polymerization of small organic molecules in solution. Specifically, using glutaraldehyde for protein crosslinking, we observe the formation of protein-rich liquid droplets for model proteins/enzymes. These droplets evolve into crosslinked microspheres. If the aqueous solutions of the protein monomer do not show LLPS at temperatures lower than the oligomerization temperature, protein-rich droplets are not observed. We experimentally link the formation of these droplets to the increase of LLPS temperature during protein oligomerization. When macroscopic aggregation competes with LLPS, a rationale choice of pH, polyethylene glycol (PEG) and salt concentrations can be used to favor LLPS relative to aggregation. Although glutaraldehyde has been extensively used to crosslink protein molecules, to our knowledge, its use in homogeneous aqueous solutions to induce LLPS has not been previously described. This work contributes to the fundamental understanding on both phase transitions of enzyme solutions and the morphology of protein condensed phases. It also provides guidance for the development of new methods based on mild experimental conditions for the preparation of enzyme-based materials. This work has been published in Langmuir, 24, 2799-2807 (2008).
PEG is a hydrophilic non-ionic polymer used together with salts for enzyme precipitation from solution. We characterize ternary diffusion in PEG-KCl-water ternary system using precision Rayleigh interferometry. The four ternary diffusion coefficients are examined and used to determine thermodynamic preferential-interaction coefficients. We find that PEG preferential hydration in the presence of salt is very large at low salt concentration. Moreover, we observe that PEG preferential hydration significantly decreases as salt concentration increases, and attribute this behavior to the polymer collapse. This indicates that the effect of PEG on enzyme precipitation decreases as salt concentration increases. Because PEG polydispersity adds complexity to the meaning of these measured diffusion coefficients, we examine important issues of polydispersity regarding the diffusion measurements and report novel equations for the extraction of diffusion moments from the Rayleigh interferometric pattern. These moments are used to define polydispersity parameters for macromolecular systems. This work has been published in J. Phys. Chem. B, 112, 4967-4974 (2008) and J. Phys. Chem. B, 112, 3633-3643 (2008).
Understanding protein solubility is important for a rational design of the conditions for the preparation of cross-linked enzyme crystals. We have reported measurements of lysozyme solubility in aqueous solutions as a function of NaCl, KCl and NH4Cl concentrations at 25 ºC and pH 4.5. Our solubility results are directly compared to preferential-interaction coefficients of these ternary solutions determined in the same experimental conditions by ternary diffusion. This comparison has provided new important insight on the dependence of protein solubility on salt concentration. We remark that the dependence of preferential-interaction coefficient as a function of salt concentration is substantially shaped by the common-ion effect. This effect plays a crucial role also on the observed behavior of lysozyme solubility. We find that the dependence of solubility on salt type and concentration strongly correlates with the corresponding dependence of the preferential-interaction coefficient. Examination of both preferential-interaction coefficients and second virial coefficients has allowed us to demonstrate that the solubility dependence on salt concentration is substantially affected by the corresponding change of protein chemical potential in the crystalline phase. We propose a simple model for the crystalline phase based on salt partitioning between solution and the hydrated protein crystal. A novel solubility equation is reported that quantitatively explain the observed experimental dependence of protein solubility on salt concentration. This work has been published on J. Am. Chem. Soc., 130, 13347-13352 (2008).
Coupled diffusion is observed in multicomponent liquid mixtures in which phase transitions may occur. This phenomenon is described by a matrix of multicomponent diffusion coefficients. We report a theoretical analysis on the role of solvation on the diffusion matrix relevant to ternary mixtures containing macromolecules or colloidal particles in the presence of salting-out conditions. A new model based on frictional coefficients between solvated solutes is reported. This model is consistent with PEG-KCl-water diffusion results. We conclude that ternary diffusion can be used to probe enzyme solvation in multicomponent mixtures. This is important not only for understanding phase transitions in enzyme aqueous mixtures but may provide guidance for understanding the effect of additives on enzymatic activity. This work has been published in J. Phys. Chem. B, 112, 4967-4974 (2008).
