Production and purification of the isolated family 2a carbohydrate-binding module from Cellulomonas fimi.
Cellulose is the most abundant polymer on Earth and in recent years, renewed interest has developed in its use for the production of biofuels and other value added products. Cellulose is degraded to glucose by the concerted action of cellulolytic enzymes that include cellulases, cellobiohydrolases, and beta-glucosidases. In many cases, these enzymes are multi-modular, being comprised of distinct catalytic and carbohydrate-binding modules. The latter appear to aid in both the adsorption of the enzymes to the insoluble cellulose substrate and the destabilization of the hydrogen-bonding network within the crystalline substrate. To better understand these dynamic processes, we have engineered a carbohydrate-binding module that can be attached to the probe of an atomic force microscope. Thus, the coding sequence for the leader peptide and carbohydrate-binding module from the Cellulomonas fimi cellulase A (cenA) was cloned and over-expressed in Escherichia coli. Site-directed mutagenesis was used to replace Thr87 of this module with Cys to facilitate covalent binding of the module to gold-plated AFM probes. The recombinant proteins with cleavable N-terminal His-tags were purified to apparent homogeneity by a combination of affinity and anion-exchange chromatographies using Ni(2+)-NTA-agarose and Source Q, respectively. Their ability to bind insoluble cellulose was demonstrated using a cellulose-binding assay involving the micro-crystalline cellulose, Avicel.
Jing
Haiqiang
H
Basic Medical College, Zhengzhou University, Zhengzhou, China.
Cockburn
Darrell
D
Zhang
Qinxian
Q
Clarke
Anthony J
AJ
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001483:Base Sequence; D001665:Binding Sites; D055592:Biophysical Phenomena; D002480:Cellulase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D040143:Cellulomonas / Q000378:metabolism*; D002482:Cellulose / Q000737:chemistry / Q000378:metabolism*; D003545:Cysteine / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism
D011994:Recombinant Proteins; D002482:Cellulose; D002480:Cellulase; D003545:Cysteine