Characterization of a multidomain cellulase from an extremely thermophilic anaerobe strain NA10

Katsuhide Miyake, Yuichi Machida, Kouji Hattori, Shinji Iijima

Research output: Contribution to journalArticle

1 Scopus citations

Abstract

The nucleotide sequence of a β-glucanase gene from an extremely thermophilic anaerobe NA10 was determined. The open reading frame extended over 3000 bp and encoded a polypeptide with a molecular mass of 113 kDa. The deduced amino acid sequence of this protein exhibited high homology to a bifunctional cellulase CelB of Caldocellum saccharolyticum. Based on the homology to GelB, the NA10 β-glucanase appears to comprise three domains: N- terminal, central, and C-terminal domains. Among these, N- and C-terminal domains apper to be catalytic domains, and the central domain to be a cellulose binding domain. These domains were joined with each other by proline and threonine rich segments (PT box). The GST-fused C-terminal domain showed CMCase and MUCase activities, but the activities of the GST-fused N- terminal domain were very weak. Zymogram analysis revealed that recombinant Escherichia coil containing the β-glucanase gene produced a protein with a molecular mass of approximately 113 kDa, which was in good agreement with that deduced from the DNA sequence. However, Western blot analysis indicated that the amount of this full length protein was very small. Several smaller abundant proteins which exhibited CMCase activity were also detected. Northern blot analysis indicated that there appear to be putative internal transcriptional initiation sites. Generation of small molecular mass species appear to be due to alternative transcription and translation from the initiation sites within the gene, or partial proteolysis.

Original languageEnglish (US)
Pages (from-to)289-296
Number of pages8
JournalJournal of Fermentation and Bioengineering
Volume85
Issue number3
DOIs
StatePublished - Jan 1 1998

Keywords

  • Endoglucanase
  • Multidomain cellulase
  • Thermophile

ASJC Scopus subject areas

  • Biotechnology
  • Applied Microbiology and Biotechnology

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