title: Enhanced soluble expression of a thermostable cellulase from Clostridium thermocellum in Escherichia coli.
abstract: In this study, the cellulase gene celD from Clostridium thermocellum was cloned into expression vectors pET-20b(+) and pHsh. While high expression can be achieved by means of both these expression systems, only the pHsh expression system gives soluble proteins. By weakening the mRNA secondary structure and replacing the rare codons for the N-terminal amino acids of the target protein, the expression level of CelD was increased from 4.1 ± 0.3 to 6.4 ± 0.4 U ml(-1) in LB medium. Recombinant CelD was purified by heat treatment followed by Ni-NTA affinity. The purified CelD exhibited the highest activity at pH 5.4 and 60°C, and retained more than 50% activity after incubation at 70°C for 1 h. The cellulase activity of CelD was significantly enhanced by Ca(2+) but inhibited by EDTA. The favorable properties of CelD offer the potential for genetic modification of strains for biomass degradation. Presently, one of the major bottlenecks for industrial cellulase users is the high cost of enzyme production. The high level expression of soluble enzymes from the pHsh expression system offers a novel approach for the production of cellulases to be used in various agro-industrial processes such as chemical, food and textile.
authors.affiliation: Research Center for Biotechnology and Biomass Energy, Nanjing Normal University, Nanjing 210046, Jiangsu, People's Republic of China.
authors.forename: Jingjing
authors.initials: J
authors.lastname: Peng
authors.forename: Weiwei
authors.initials: W
authors.lastname: Wang
authors.forename: Yuyao
authors.initials: Y
authors.lastname: Jiang
authors.forename: Mingjie
authors.initials: M
authors.lastname: Liu
authors.forename: Hui
authors.initials: H
authors.lastname: Zhang
authors.forename: Weilan
authors.initials: W
authors.lastname: Shao
mesh_terms: D002480:Cellulase / Q000096:biosynthesis* / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods*; D048013:Acetivibrio thermocellus / Q000201:enzymology* / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D007218:Industrial Microbiology / Q000379:methods*; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction
chemical_list: D004269:DNA, Bacterial; C087879:endoglucanase CelD; D002480:Cellulase