title: Enzymatic hydrolysis of cellulose by the cellobiohydrolase domain of CelB from the hyperthermophilic bacterium Caldicellulosiruptor saccharolyticus.
abstract: The celB gene of Caldicellulosiruptor saccharolyticus was cloned and expressed in Escherichia coli to create a recombinant biocatalyst for hydrolyzing lignocellulosic biomass at high temperature. The GH5 domain of CelB hydrolyzed 4-nitrophenyl-β-D-cellobioside and carboxymethyl cellulose with optimum activity at pH 4.7-5.5 and 80°C. The recombinant GH5 and CBM3-GH5 constructs were both stable at 80°C with half-lives of 23 h and 39 h, respectively, and retained >94% activity after 48 h at 70°C. Enzymatic hydrolysis of corn stover and cellulose pretreated with the ionic liquid 1-ethyl-3-methylimidazolium acetate showed that GH5 and CBM3-GH5 primarily produce cellobiose, with product yields for CBM3-GH5 being 1.2- to 2-fold higher than those for GH5. Confocal microscopy of bound protein on cellulose confirmed tighter binding of CBM3-GH5 to cellulose than GH5, indicating that the enhancement of enzymatic activity on solid substrates may be due to the substrate binding activity of CBM3 domain.
authors.affiliation: Deconstruction Division, Joint BioEnergy Institute, Emeryville, CA 94608, USA.
authors.forename: Joshua I
authors.initials: JI
authors.lastname: Park
authors.forename: Michael S
authors.initials: MS
authors.lastname: Kent
authors.forename: Supratim
authors.initials: S
authors.lastname: Datta
authors.forename: Bradley M
authors.initials: BM
authors.lastname: Holmes
authors.forename: Zhaohua
authors.initials: Z
authors.lastname: Huang
authors.forename: Blake A
authors.initials: BA
authors.lastname: Simmons
authors.forename: Kenneth L
authors.initials: KL
authors.lastname: Sale
authors.forename: Rajat
authors.initials: R
authors.lastname: Sapra
mesh_terms: D002482:Cellulose / Q000378:metabolism*; D043366:Cellulose 1,4-beta-Cellobiosidase / Q000737:chemistry / Q000378:metabolism*; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D006868:Hydrolysis; D018613:Microscopy, Confocal; D046968:Thermoanaerobacter / Q000201:enzymology*
chemical_list: D002482:Cellulose; D043366:Cellulose 1,4-beta-Cellobiosidase