Crystallization studies on glucose isomerase (2000)

Proteins are biological macromolecules that have an increasing importance in foodnprocessing and in therapeutic products. Industrial crystallization for purification andnseparation of proteins has only been seriously considered during the past decade. Itnhas many process parallels with small molecule crystallization, including anremarkable ability to purify proteins. Moreover, crystallization processes are distinctlyncheaper and simpler to operate than traditional adsorption technologies, which arencurrently used to purify proteins. Overall, this thesis aimed to extend the engineeringnresearch knowledge of industrial bulk crystallization processes for proteins. Thenenzyme glucose isomerase (GI), which is used in the manufacture of high fructosencorn syrups (HFCS), was studied in this work. The broad topics of study included thenassessment and miniaturisation of experimental techniques, the solubility phasendiagram, crystal growth and nucleation kinetics and graphoepitaxial crystallization.n n To design a bulk crystallization process, kinetic data are required for the crystalngrowth and nucleation relationships with processing variables. The most importantnvariables are commonly temperature and solute concentration. The crystal growthnkinetics of GI were measured as a function of temperature (15, 20 and 25 dC) andnconcentration (2 -53 mg/mL) at pH 7.0 in 10% (w/v) ammonium sulphate solutions.nThis was performed using a miniaturised bulk crystallization technique that wasndeveloped in this work. Under equivalent conditions, the growth kinetics were alsonstudied by the static technique. The static technique is more commonly used innfundamental studies of protein crystal growth. However, the growth kinetics of GInwere found to be significantly different when measured by both techniques. In thenagitated bulk crystallizer, GI demonstrated a typical second order growth dependencenon supersaturation and a remarkably high dependence of growth rate on temperature.nThis was characterised as an activation energy of 179 p 4 kJ/mol. In the static system,nGI demonstrated a 3.1 growth order and an even higher activation energy ofn280 p 12 kJ/rnol. Moreover, static crystals had slower growth rates under equivalentnconditions, with more than an order of magnitude difference at the lower supersatrations tested. Cold crystallization conditions (l 5 dC) are predicted as thenconditions to maximise the growth rates of GI (up to about 5 mm per minute).nHowever, the incorrect kinetics found by the static technique caused the predictedngrowth rates at these temperatures to be an order of magnitude greater than thosenpredicted by bulk crystallization kinetics. Therefore industrially aimed researchnshould measure growth kinetics by bulk crystallization techniques.n n The miniaturised bulk crystallization technique that was developed only requirednabout 1 gram of protein per experiment. This technique would enable kinetic studiesnin situations where a protein is scarce or costly. This was only possible by usingnimage analysis to measure crystal size distributions. An improved means of datananalysis was incorporated into the technique that enabled simultaneous measurementnof crystal growth and nucleation kinetics from batch crystallizations. Simultaneousnstudy of nucleation kinetics was necessary because the metastable zone of GI wasnvery small (a= 2.5), resulting in a remarkably high rate of nucleation during batchncrystallizations. The nucleation data was correlated with a new model for nucleationnin batch crystallizations. This model pieces together the solute aggregation andncollision sources of nuclei from a knowledge of the nucleation thresholds. Thenparameters suggested that collision nucleation was the key issue for GI and wouldnbecome a greater problem as the crystal content increases.n n A model for the approach to equilibrium solubility was developed. This found that thenusual waiting period of a day or a few weeks for a solution to reach solubilitynequilibrium by crystallization is usually not long enough for proteins. The criticalnimportance of accurate solubility data for the analysis of crystal growth kinetics wasndemonstrated. A solubility error can greatly affect the estimates of growth order andngrowth constant. This may explain some of the high growth orders (n g 3) found fornseveral other proteins in the literature. To improve the accuracy and speed ofnsolubility data generation, the approach to equilibrium model was adapted for curvenfitting purposes in order to fit dynamic concentration data and thereby extrapolate it tonthe equilibrium solubility value.n n Graphoepitaxial crystallization of GI was trailed using highly oriented frictionndeposited PTFE substrates. Oriented protein crystals could present new applications.nHowever, no significant orientation of GI crystals was found on the PTFE substrates.nGraphoepitaxial substrates with greater groove width and period than that for PTFEnmay induce macromolecular crystal orientation. n Observations on the GI crystal habit found that, unlike lysozyme, GI was insensitivento the variables of temperature and concentration. However, other species present innsolution, such as the precipitant, appeared to control the crystal habit.n n This work has extended the knowledge of protein crystallization for industrialnpurposes. The static technique was found to be a misleading way to study crystalngrowth kinetics of GI. In its place, a new miniaturised bulk technique was used thatnenabled simultaneous analysis of growth and nucleation kinetics from batchnexperiments. Correlation s for the solubility, growth and nucleation kinetics of GI werenmade. Further experimental work on GI is recommended to give more data for thenparameters of the nucleation model. This would provide the data to support a processnmodelling and optimisation study.n