Characterization and crystallization of the Endoglucanase A from Clostridium Cellulolyticum in solution

This study deals with the Endoglucanase of the family A (CelCCA) from Clost ridium Cellulolyticum (MW = 43 kDa, pI congruent to 6). CelCCA takes part i n a multienzyme complex (cellulosome) including a majority of cellulases wh ose detailed molecular organization and mode of action are not well known. The aim of this work is to understand the crystal growth mechanisms of CelC CA. Solubilities and crystal growth rates were measured, and the behavior o f CelCCA molecules in under and supersaturated solutions was investigated. The presence of different isoenzymes in the CelCCA solution was observed by mass spectroscopy and the presence of aggregates was detected by dynamic l ight scattering. It was shown that these aggregates, due to the different i soenzymes of CelCCA, hinder the crystallization of CelCCA. Interactions bet ween protein molecules, measured by small-angle X-ray scattering, were foun d attractive under crystallization conditions (14% PEG 4000, 90 mM sodium c itrate, pH 6). The addition of CaCl2 to the solution was found to improve t he crystallization, favoring the salting-out effect, but had no influence o n the solubility and on the structure of CelCCA. A new polymorph of CelCCA was found, and the solubility of the two polymorphs was measured. The two p olymorphs, which grew under similar conditions, differ mainly by their crys tallographic b parameter, the space group P2(1)2(1)2(1) being conserved. Gr owth rates were measured, it was found that the more soluble polymorph has a higher growth rate. (C) 1999 Elsevier Science B.V. All rights reserved.