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

Abstract This study deals with the Endoglucanase of the family A (CelCCA) from Clostridium Cellulolyticum (MW=43 kDa, pI≅6). CelCCA takes part in a multienzyme complex (cellulosome) including a majority of cellulases whose detailed molecular organization and mode of action are not well known. The aim of this work is to understand the crystal growth mechanisms of CelCCA. Solubilities and crystal growth rates were measured, and the behavior of 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 light scattering. It was shown that these aggregates, due to the different isoenzymes of CelCCA, hinder the crystallization of CelCCA. Interactions between protein molecules, measured by small-angle X-ray scattering, were found attractive under crystallization conditions (14% PEG 4000, 90 mM sodium citrate, pH 6). The addition of CaCl 2 to the solution was found to improve the crystallization, favoring the salting-out effect, but had no influence on 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 polymorphs, which grew under similar conditions, differ mainly by their crystallographic b parameter, the space group P2 1 2 1 2 1 being conserved. Growth rates were measured, it was found that the more soluble polymorph has a higher growth rate.