Sequencing and expression of a cellodextrinase (ced1) gene from Butyrivibrio fibrisolvens H17c cloned in Escherichia coli.
The nucleotide sequence of a 2.314 kb DNA segment containing a gene (ced1) 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. cellulosa endoglucanase (EG), and a cellulase from the avocado fruit (Persea americana). The ced1 gene product Ced1 showed cellodextrinase activity and rapidly hydrolysed short-chain cellodextrins to yield either cellobiose or cellobiose and glucose as end products. The Ced1 enzyme released cellobiose from p-nitrophenyl-beta-D-cellobioside and the enzyme was not inhibited by methylcellulose, an inhibitor of endoglucanase activity. Although the major activity of the Ced1 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 Mr of approximately 61,000. The calculated Mr of the ced1 gene product was 61,023.
Berger
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Department of Microbiology, University of Cape Town, Rondebosch, South Africa.
Jones
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Jones
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Woods
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D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001438:Bacteroidaceae / Q000201:enzymology / Q000235:genetics*; D001483:Base Sequence; D002480:Cellulase / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D016384:Consensus Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D013379:Substrate Specificity
D001426:Bacterial Proteins; C066565:ced1 protein, Butyrivibrio fibrisolvens; D002480:Cellulase