title: Optimization of affinity digestion for the isolation of cellulosomes from Clostridium thermocellum.
abstract: The affinity digestion process for cellulase purification consisting of binding to amorphous cellulose, and amorphous cellulose hydrolysis in the presence of dialysis (Morag et al., 1991), was optimized to obtain high activity recoveries and consistent protein recoveries in the isolation of Clostridium thermocellum cellulase. Experiments were conducted using crude supernatant prepared from C. thermocellum grown on either Avicel or cellobiose. While no difference was observed between Avicel-grown or cellobiose-grown cellulase in the adsorption step, differences were observed during the hydrolysis step. The optimal amorphous cellulose loading was found to be 3 mg amorphous cellulose per milligram supernatant protein. At this loading, 90-100% of activity in the crude supernatant was adsorbed. Twenty-four-hour incubation with the amorphous cellulose during the adsorption stage was found to result in maximal and stable adsorption of activity to the substrate. By fitting the adsorption data to the Langmuir model, an adsorption constant of 410 L/g and a binding capacity of 0.249 g cellulase/g cellulose were obtained. The optimal length of time for hydrolysis was found to be 3 hr for cellulase purified from Avicel cultures and 4 hr for cellulase purified from cellobiose cultures. These loadings and incubation times allowed for more than 85% activity recovery.
authors.affiliation: a Thayer School of Engineering, Dartmouth College , Hanover , New Hampshire , USA.
authors.forename: Lois A
authors.initials: LA
authors.lastname: St Brice
authors.forename: Xiongjun
authors.initials: X
authors.lastname: Shao
authors.forename: Javier A
authors.initials: JA
authors.lastname: Izquierdo
authors.forename: Lee R
authors.initials: LR
authors.lastname: Lynd
mesh_terms: D000327:Adsorption; D044603:Cellulosomes / Q000378:metabolism*; D002846:Chromatography, Affinity / Q000379:methods*; D048013:Acetivibrio thermocellus / Q000254:growth & development / Q000378:metabolism*; D006868:Hydrolysis