title: Biochemical and mutational analyses of a multidomain cellulase/mannanase from Caldicellulosiruptor bescii.
abstract: Thermophilic cellulases and hemicellulases are of significant interest to the biofuel industry due to their perceived advantages over their mesophilic counterparts. We describe here biochemical and mutational analyses of Caldicellulosiruptor bescii Cel9B/Man5A (CbCel9B/Man5A), a highly thermophilic enzyme. As one of the highly secreted proteins of C. bescii, the enzyme is likely to be critical to nutrient acquisition by the bacterium. CbCel9B/Man5A is a modular protein composed of three carbohydrate-binding modules flanked at the N terminus and the C terminus by a glycoside hydrolase family 9 (GH9) module and a GH5 module, respectively. Based on truncational analysis of the polypeptide, the cellulase and mannanase activities within CbCel9B/Man5A were assigned to the N- and C-terminal modules, respectively. CbCel9B/Man5A and its truncational mutants, in general, exhibited a pH optimum of ∼5.5 and a temperature optimum of 85°C. However, at this temperature, thermostability was very low. After 24 h of incubation at 75°C, the wild-type protein maintained 43% activity, whereas a truncated mutant, TM1, maintained 75% activity. The catalytic efficiency with phosphoric acid swollen cellulose as a substrate for the wild-type protein was 7.2 s(-1) ml/mg, and deleting the GH5 module led to a mutant (TM1) with a 2-fold increase in this kinetic parameter. Deletion of the GH9 module also increased the apparent k(cat) of the truncated mutant TM5 on several mannan-based substrates; however, a concomitant increase in the K(m) led to a decrease in the catalytic efficiencies on all substrates. These observations lead us to postulate that the two catalytic activities are coupled in the polypeptide.
authors.affiliation: Energy Biosciences Institute, University of Illinois, Urbana, Illinois, USA.
authors.forename: Xiaoyun
authors.initials: X
authors.lastname: Su
authors.forename: Roderick I
authors.initials: RI
authors.lastname: Mackie
authors.forename: Isaac K O
authors.initials: IK
authors.lastname: Cann
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002480:Cellulase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002482:Cellulose / Q000378:metabolism; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D004795:Enzyme Stability; D006094:Gram-Positive Bacteria / Q000201:enzymology* / Q000235:genetics; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008351:Mannans / Q000737:chemistry / Q000378:metabolism; D008361:Mannosidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D017422:Sequence Analysis, DNA; D013696:Temperature; D044902:beta-Mannosidase / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D008351:Mannans; D002482:Cellulose; D008361:Mannosidases; D044902:beta-Mannosidase; D002480:Cellulase