Isolation and characterization of genes encoding thermoactive and thermostable dextranases from two thermotolerant soil bacteria

Thermotolerant Paenibacillus strain Dex70-1B and unidentified strain Dex70-34 produce thermoactive dextran-degrading enzymes. Plasmid-based genomic DNA libraries constructed from mixed bacterial cultures containing Dex70-1B or Dex70-34 were screened for the ability to confer dextranolytic activity a...

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Bibliographic Details
Published in:Current microbiology 2004-11, Vol.49 (5), p.327-333
Main Authors: Finnegan, Patrick M, Brumbley, Stevens M, O'Shea, Michael G, Nevalainen, Helena, Bergquist, Peter L
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Amino acids
Bacteria
Bacteria - enzymology
Bacteria - genetics
Bacteria - isolation & purification
Base Sequence
Dextranase - genetics
Dextranase - metabolism
DNA, Bacterial - genetics
E coli
Enzyme Stability
Escherichia coli
Escherichia coli - enzymology
Escherichia coli - genetics
Ethanol
Genes, Bacterial
Hot Temperature
Hydrogen-Ion Concentration
Kinetics
Microbiology
Molecular Sequence Data
Open Reading Frames
Paenibacillus
Phylogeny
Recombinant Proteins - genetics
Recombinant Proteins - metabolism
Sequence Homology, Amino Acid
Soil Microbiology
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Summary:Thermotolerant Paenibacillus strain Dex70-1B and unidentified strain Dex70-34 produce thermoactive dextran-degrading enzymes. Plasmid-based genomic DNA libraries constructed from mixed bacterial cultures containing Dex70-1B or Dex70-34 were screened for the ability to confer dextranolytic activity at 70 degrees C onto Escherichia coli. One gene, designated dex1, was isolated from each strain. The Dex70-1B and Dex70-34 dex1 gene sequences were non-identical, and encoded proteins containing 597 (M(r) 68.6 kDa) and 600 amino acids (M(r) 69.2 kDa), respectively. The Dex1 amino acid sequences were most similar to one another, and formed a new clade among the family 66 glycosyl hydrolase sequences. Expression of the Dex1 proteins in E. coli produced dextranolytic activity that converted ethanol-insoluble blue dextran into an ethanol-soluble form, suggestive of endodextranases (EC 3.2.1.11). Both enzymes were most active at about 60 degrees C and pH 5.5, and retained more than 70% maximal activity after incubation at 57 degrees C for 9.5 h in the absence of substrate.
ISSN:0343-8651
1432-0991
DOI:10.1007/s00284-004-4308-5