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Volume 142, Issue 4

Molecular study and overexpression of the cellulase gene in Free

Abstract

The CelF-encoding sequence was isolated from genomic DNA using the inverse PCR technique. The gene lies between (the gene encoding the cellulosome scaffolding protein) and (coding for the endoglucanase C) in the large cluster of this mesophilic cellulolytic species. Comparisons between the deduced amino acid sequence of the mature CelF (693 amino acids, molecular mass 77626) and those of other ß-glycanases showed that this enzyme belongs to the recently proposed family L of cellulases (family 48 of glycosyl hydrolases). The protein was overproduced in using the T7 expression system. It formed both cytoplasmic and periplasmic inclusion bodies when induction was performed at 37 °. Surprisingly, the protein synthesized from the cytoplasmic production vector was degraded in the protease-deficient strain BL21(DE3). The induction conditions were optimized with regard to the concentration of inductor, cell density, and temperature and time of induction in order to overproduce an active periplasmic protein (CelFp) which was both soluble and stable. It was collected using the osmotic shock method. The enzymic degradation of various cellulosic substrates by CelFp was studied. CelFp degraded swollen Avicel more efficiently than substituted soluble CM-cellulose or crystalline Avicel and was not active on xylan. Its activity is therefore quite different from that of endoglucanases, which are most active on CM-cellulose.

  • Received:
  • Accepted:
  • Revised:
  • Published Online:
Keyword(s): cellulase , Clostridium cellulolyticum , overproduction and periplasmic inclusion bodies
© Society for General Microbiology 1996
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1996-04-01
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Molecular study and overexpression of the Clostridium cellulolyticum celF cellulase gene in Escherichia coli
Microbiology 142, 1013 (1996); https://doi.org/10.1099/00221287-142-4-1013
/content/journal/micro/10.1099/00221287-142-4-1013
/content/journal/micro/10.1099/00221287-142-4-1013
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