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 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.
Finnegan
Patrick M
PM
Department of Biological Sciences, Macquarie University, Sydney, NSW, Australia.
Brumbley
Stevens M
SM
O'Shea
Michael G
MG
Nevalainen
Helena
H
Bergquist
Peter L
PL
D000595:Amino Acid Sequence; D001419:Bacteria / Q000201:enzymology* / Q000235:genetics* / Q000302:isolation & purification; D001483:Base Sequence; D004269:DNA, Bacterial / Q000235:genetics; D003910:Dextranase / Q000235:genetics* / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005798:Genes, Bacterial; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010802:Phylogeny; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012988:Soil Microbiology
D004269:DNA, Bacterial; D011994:Recombinant Proteins; D003910:Dextranase