Affiliation: | a Department of Bioapplied Chemistry, Osaka City University, Sugimoto 3-3-138, Sumiyoshi-ku, Osaka 558, Japan b Engineering Research Laboratories, Kaneka Corporation, 1-8, Miyamae-machi, Takasago-cho, Takasago-shi, Hyogo 676, Japan |
Abstract: | The mechanism of crystallization of enzyme protein thermolysin was investigated. The size distribution of thermolysin precipitates was measured by dynamic light scattering during precipitation, and the surface and cross section of the finally obtained precipitate were observed by scanning electron microscopy. The thermolysin precipitates obtained at the initial supersaturation ratio of 8.9 to 164 and pH 7.0 were hexagonal rods having an average size of 9.2×1.5 μm, and were composed of a number of small particles of 15 to 200 nm in diameter. The average size of the small particles was 60 nm in diameter, and the formation of the particles was found to be completed in the early stage of precipitation. Observation of the finally recovered thermolysin precipitate by polarizing microscopy revealed that the precipitate is a crystalline solid. From these data, a possible mechanism of thermolysin crystallization was proposed. The crystallization proceeds through two steps: the first step is the formation of primary particles, and the second step is crystal growth by highly ordered aggregation of the primary particles. |