Production and purification of the isolated family 2a carbohydrate-binding module from Cellulomonas fimi

Haiqiang Jing, Darrell Cockburn, Qinxian Zhang, Anthony J. Clarke

Research output: Contribution to journalArticlepeer-review

4 Scopus citations

Abstract

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 β-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 Ni2+-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.

Original languageEnglish (US)
Pages (from-to)63-68
Number of pages6
JournalProtein Expression and Purification
Volume64
Issue number1
DOIs
StatePublished - Mar 2009

All Science Journal Classification (ASJC) codes

  • Biotechnology

Fingerprint Dive into the research topics of 'Production and purification of the isolated family 2a carbohydrate-binding module from Cellulomonas fimi'. Together they form a unique fingerprint.

Cite this