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Sequencing and expression of a cellodextrinase (ced1) gene from Butyrivibrio fibrisolvens H17c cloned in Escherichia coli

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Summary

The nucleotide sequence of a 2.314 kb DNA segment containing a gene (cedl) expressing cellodextrinase activity from Butyrivibrio fibrisolvens H17c was determined. The B. fibrisolvens H17c gene was expressed from a weak internal promoter in Escherichia coli and a putative consensus promoter sequence was identified upstream of a ribosome binding site and a GTG start codon. The complete amino acid sequence (547 residues) was deduced and homology was demonstrated with the Clostridium thermocellum endoglucanase D (EGD), Pseudomonas fluorescens var. cellulose endoglucanase (EG), and a cellulase from the avocado fruit (Persea americana). The ced1 gene product Cedl showed cellodextrinase activity and rapidly hydrolysed short-chain cellodextrins to yield either cellobiose or cellobiose and glucose as end products. The Cedl enzyme released cellobiose from p-nitrophenyl-β-d-cellobioside and the enzyme was not inhibited by methylcellulose, an inhibitor of endoglucanase activity. Although the major activity of the Cedl enzyme was that of a cellodextrinase it also showed limited activity against endoglucanase specific substrates [carboxymethylcellulose (CMC), lichenan, laminarin and xylan]. Analysis by SDS-polyacrylamide gel electrophoresis with incorporated CMC showed a major activity band with an apparent M r of approximately 61000. The calculated M r of the ced1 gene product was 61023.

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Abbreviations

Ap:

ampicillin

ced1 :

gene coding for Ced1

Ced1:

cellodextrinase from B. fibrisolvens

CMC:

carboxymethylcellulose

LB:

Luria Bertani

ORF:

open reading frame

pNPC:

p-nitrophenyl-β-d-cellobioside

PC:

phosphate citrate

HCA:

hydrophobic cluster analysis

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Authors and Affiliations

  1. Department of Microbiology, University of Cape Town, 7700, P B Rondebosch, South Africa

    Eldie Berger & David R. Woods

  2. Department of Microbiology, University of Otago, P O Box 56, Dunedin, New Zealand

    Winsome A. Jones & David T. Jones

Authors
  1. Eldie Berger
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  2. Winsome A. Jones
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  3. David T. Jones
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  4. David R. Woods
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Communicated by H. Hennecke

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Berger, E., Jones, W.A., Jones, D.T. et al. Sequencing and expression of a cellodextrinase (ced1) gene from Butyrivibrio fibrisolvens H17c cloned in Escherichia coli . Molec. Gen. Genet. 223, 310–318 (1990). https://doi.org/10.1007/BF00265068

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  • DOI: https://doi.org/10.1007/BF00265068

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