ISSN:
1432-0614
Keywords:
Clostridium thermocellum
;
Cellulasex
;
Cellulosome
;
CelS
;
Exoglucanase
Source:
Springer Online Journal Archives 1860-2000
Topics:
Biology
,
Process Engineering, Biotechnology, Nutrition Technology
Notes:
Abstract Clostridium thermocellum ATCC 27405 produces an extremely complicated multi-component cellulase aggregate (cellulosome) highly active on crystalline cellulose. From the cellulosome, two subunits, CelS (or S s ;M r = 82 000) and CelL (or S l , CipA;M r = 250 000), have been identified as essential for crystalline cellulose degradation [Wu et al. (1988) Biochemistry 27:1703]. We have determined the DNA sequence of thecelS gene from four cloned DNA fragments encompassing this gene [Wang et al. (1993) J Bacteriol 175:1293]. To express the entirecelS gene inEscherichia coli, thecelS structural gene was amplified by the polymerase chain reaction (PCR) employing the PCR primers corresponding to sequences flanking the desired gene. This PCR product (2.1 x 103 bases; 2.1 kb) was cloned into anE. coli expression vector pRSET B. Subsequent expression of the cloned gene resulted in a fusion protein (rCelS;M r = 86 000) as inclusion bodies. The rCelS protein was recognized specifically by an anti-CelS antiserum in a Western blot analysis. The inclusion bodies were purified and solubilized in 5m urea. The refolded rCelS produced very little reducing sugar from carboxymethylcellulose. However, it showed a higher activity on the crystalline cellulose (Avicel) and an even higher activity on phosphoricacid-swollen Avicel. These results indicate that the CelS is an exoglucanase.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1007/BF00902740
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