Advances in Protein Precipitation

Abstract

Proteins are biological macromolecules, which are among the key components of all living organisms. Proteins are nowadays present in all fields of biotech industry, such as food and feed, synthetic and pharmaceutical industry. They are isolated from their natural sources or produced in different cell lines. Most important large-scale bioseparation techniques for proteins are precipitation, crystallization, membrane separations and chromatography. Food and feed proteins are usually recovered using low-resolution techniques such as membrane filtration and precipitation, whereas high-value proteins are recovered and purified by means of high-resolution techniques, such as crystallization and chromatography. Precipitation is a mature technique, which enables crude recovery of proteins from natural sources. Industrial scale precipitation is often associated with the consumption of large amounts of auxiliary materials, such as salts or organic solvents, and hence regarded as a burden for the environment. This thesis presents advances in protein precipitation based on a novel method, CO2-aided acidification. Precipitation based on carbon dioxide is an environmentally friendly technique, which relies on protein precipitation at its isoelectric point. The CO2-based acidification is used to develop a novel method for protein purification. It also forms a basis of a newly developed technique for active ingredient immobilization. Nowadays precipitation of food proteins is mainly performed using precipitants such as salts or organic solvents. As a consequence the precipitate needs to be treated to remove the precipitant and all process streams have to be additionally processed before the disposal. This puts an additional burden on the environment and the energy consumption. Chapter 2 presents a novel method for purification of glycinin from soy by using a single precipitant, CO2. The gaseous CO2 is used to acidify the solution containing a mixture of soy proteins, using the pressure as the control parameter. The subtle acidification enables selective precipitation of glycinin from the solution. The selective precipitation of glycinin is repeated several times to obtain a high grade of purity (98%). The protein is subsequently freeze–dried and a later re-solubilization indicates that the protein did not denature during the purification process. The purification process was successfully scaled up from a 50 mL to a 1 L vessel. The characteristic time analysis of the underlying mechanisms (precipitation, liquid mixing and mass transfer) showed that they remain unchanged with the scale-up. In addition, the characteristic time analysis reveals that the mass-transfer of the carbon dioxide from the gaseous to the liquid phase is the rate-limiting step. Bioseparation processes development traditionally relies on the empirical approach. Recently, however, a lot of attention has been devoted to the rational design of the bioseparation processes. For this approach protein characterization is a crucial step. One way to obtain valuable information about protein behavior is to measure thermodynamic properties of the protein solution. In Chapter 3 we present a study of thermodynamic properties of glycinin solutions as a function of pH. The osmotic second virial coefficient of glycinin solutions was measured by using static light scattering (SLS). Glycinin proved to be a cumbersome molecule for the SLS measurements, due to its size and pH-dependent solubility. We established a working window and successfully performed static and dynamic light scattering of glycinin solutions. The SLS measurements revealed that the second osmotic virial coefficient becomes highly negative with decreasing pH, which corresponds to the known property of glycinin to precipitate at its isoelectric point. The CLEA technology has been extensively developed in the past decades. It is based on a two-step approach: initial aggregation followed by cross-linking. The technique has been successfully used over years to immobilize different enzymes. In Chapter 4 we present a scouting study for using carbon dioxide as a precipitant in the initial aggregation step. The CO2-aided precipitation was tested for a number of enzymes using soy protein as a control. Under the conditions tested in the scouting study no aggregation in enzyme solutions was observed when subjected to the CO2-aided acidification. Theoretical considerations on the observed phenomena are presented. In addition, a screening study of the classical organic precipitants for the manufacturing of CLEAs is described. The morphology and structure of the CLEA particles is evaluated. Immobilization of active ingredients has been widely explored for the past few decades. It is applied in food, cosmetic and pharmaceutical industry and relies on a stabile matrix to immobilize and protect an active ingredient. The purpose is to obtain prolonged activity and in some cases sustained release of the active ingredient. In Chapter 5 we present a novel method for active ingredient immobilization based on the CO2-aided co-precipitation with glycinin. Thanks to its property to precipitate easily at its isoelectric point, glycinin was used as a matrix for the active ingredient co-precipitation. The proof of principle was shown using the enzyme lipase, which is successfully co-precipitated into the glycinin matrix while retaining its activity. Importantly, the obtained co-precipitate was of food-grade quality, without organic precipitants or salts. This makes an obvious advantage in reduced post-processing and a broad range of application possibilities. In Chapter 6 a detailed characterization of the co-precipitate of glycinin and lipase is presented. The co-precipitate was tested for resolubilization and lipase activity at different pH conditions. The resolubility of the co-precipitate was shown to be equivalent to that of glycinin. The lipase activity was enhanced as compared to native lipase. In addition, lipase in the co-precipitate retained activity at otherwise harsh conditions, indicating the protective role of the glycinin matrix. The dissolution of the co-precipitate was successfully described by a model based on two mechanisms: disintegration of the particles into multimers and diffusion of the multimers into the liquid phase. The model fitted reasonably well the experimentally obtained data. Lipase activity in the liquid phase was observed even when the co-precipitate scarcely dissolved. This indicates that lipase can diffuse from the co-precipitate particles into the liquid phase.