title: Cell-free protein synthesis and substrate specificity of full-length endoglucanase CelJ (Cel9D-Cel44A), the largest multi-enzyme subunit of the Clostridium thermocellum cellulosome.
abstract: Endoglucanase CelJ (Cel9D-Cel44A) is the largest multi-enzyme subunit of the Clostridium thermocellum cellulosome and is composed of glycoside hydrolase (GH) families 9 and 44 (GH9 and GH44) and carbohydrate-binding module (CBM) families 30 and 44 (CBM30 and CBM44). The study of CelJ has been hampered by the inability to isolate full-length CelJ from recombinant Escherichia coli cells. Here, full-length CelJ and its N- and C-terminal segments, CBM30-GH9 (Cel9D) and GH44-CBM44 (Cel44A), were synthesized using a wheat germ cell-free protein synthesis system and then were purified to homogeneity. Analysis of the substrate specificities of CelJ and its derivatives demonstrated that the fusion of Cel9D and Cel44A results in threefold synergy for the degradation of xyloglucan, one of the major structural polysaccharides of plant cell walls. Because CelJ displayed broad substrate specificity including significant carboxymethylcellulase (CMCase) and xylanase activities in addition to high xyloglucanase activity, CelJ may play an important role in the degradation of plant cell walls, which are composed of highly heterogeneous polysaccharides. Furthermore, because Cel9D, but not Cel44A, acts as a semi-processive endoglucanase, the different modes of action between Cel9D and Cel44A may be responsible for the observed synergistic effect on the activity of CelJ (Cel9D-Cel44A).
authors.affiliation: Department of Chemical Biology & Applied Chemistry, College of Engineering, Nihon University, Koriyama, Fukushima, Japan. nhirano@chem.ce.nihon-u.ac.jp
authors.forename: Nobutaka
authors.initials: N
authors.lastname: Hirano
authors.forename: Hiroki
authors.initials: H
authors.lastname: Hasegawa
authors.forename: Satoshi
authors.initials: S
authors.lastname: Nihei
authors.forename: Mitsuru
authors.initials: M
authors.lastname: Haruki
mesh_terms: D002474:Cell-Free System; D002480:Cellulase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D048013:Acetivibrio thermocellus / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D009097:Multienzyme Complexes / Q000378:metabolism*; D014176:Protein Biosynthesis; D021122:Protein Subunits; D017384:Sequence Deletion; D013379:Substrate Specificity
chemical_list: D009097:Multienzyme Complexes; D021122:Protein Subunits; C473132:cellulosome, Clostridium; D002480:Cellulase