YAMASHITA Yoshiyuki Tohoku University, Department of Engineering, Associate Professor, 大学院・工学研究科, 助教授 (60200698)
IHARA Manabu Tohoku University, Institute of Multidisciplinary Research for Advanced Materials, Research Associate, 多元物質科学研究所, 助手 (90270884)
|Budget Amount *help
¥14,200,000 (Direct Cost: ¥14,200,000)
Fiscal Year 2003: ¥4,800,000 (Direct Cost: ¥4,800,000)
Fiscal Year 2002: ¥9,400,000 (Direct Cost: ¥9,400,000)
Summaries of this project are as follows.
From recent studies for the phase equilibrium of aqueous protein solutions, the possibility of a metastable liquid-liquid phase separation is discussed and some groups tried to develop theoretical models to describe such kinds of phase behavior. We thought that pressure can be used as a suitable variable to control the phase behavior of protein solutions. The development of experimental method to produce protein crystal is the aim of this project.
1 Measurements of crystal growth of protein from aqueous solutions and thermophysical properties of protein solution.
The rate of crystal growth of lysozyme from aqueous solution were measured under high pressures up to 100MPa. It was found that the growth rate strongly depends on the pressure. The thermophysical properties (viscosity, density, solubility) of lyzosyme aqueous solutions were also measured under pressure, which were needed as the parameters for theoretical analysis. The density of solution
were measured with the glass pycnometer method, the viscosity falling needle method, and the solubility from the temporal change of protein concentration in the solution. From the solubility data, we can estimate the supersaturation degree accurately. The crystal growth rate decreases with pressure increases. From the analysis of pressure dependence of crystal growth, it was found that rate determining process should be mass transfer process.
2 Measurement of production rate of crystal nucleus.
The production rate of crystal nucleus number was measured with the direct observation method under high pressures up to 100MPa, The growth rate decreases with pressure increases which is almost same as the crystal growth rate. Therefore, if lysozyme crystals were produced under high pressures, few number but much larger size crystal can be obtained, It was proved to be correct from the experiments. The rate determining process for the production of crystal nucleus was found to be mass transfer process.
3 Development of experimental method for the micro-crystal of protein.
The above 2 themes were based on the experiment using static pressure. By using static pressure, we can get large size of protein crystal. In this themes, dynamic pressure such as drastic pressure drop in nozzle was applied to promote the liquid-liquid phase separation of protein solutions, To do so, we selected the rapid expansion method with supercritical fluid solution, At first, organic compound such as naphthalene or acetyl salicylic acid were used to test the apparatus. Then, the aqueous protein solution and supercritical carbon dioxide was mixed and expanded from the nozzle. With the use of nozzles with different diameter, the aqueous solutions were expanded into the chamber over a wide range of temperature and pressure. The microcrystal of lyzosyme can be produced at the optimum conditions of nozzle and temperature and pressure conditions of chamber. From these results, we can conclude that liquid-liquid phase separation induced pressure can be occurred in the nozzle. Less