[Cloning and characterization of a large metagenomic DNA fragment containing glycosyl-hydrolase genes].
The problem of search for and characterization of enzymes synthesized by non-cultivated microorganisms is presently being settled by creating metagenomic libraries. A 6000-clone library with the average size of its inserts amounting to 15 bp has been constructed on the basis of total DNA isolated from cow rumen microorganisms. As the result of the screening of the library on plates with different substrates, a clone was selected that efficiently hydrolyzed lichenan and carboxymethylcellulose. The clone contained the recombinant plasmid pBlue-13 bearing a 12071 bp.-long metagenomic fragment carrying ten open reading frames, two of them being identified as glycosyl hydrolase genes. No homology of the metagenomic DNA with any known microorganism genomes was revealed. The amino acid sequence, deduced on the basis of frame 4 and denoted by Xyl3A, bears resemblance with beta-xylosidases of glycosyl hydrolase Family 3. Frame 6 encodes polypeptide Cel5A homologous to cellulases of glycosyl hydrolase Family 5. The amino acid sequences deduced on the basis of seven out of ten open reading frames were homologous to proteins of microorganisms belonging to the Bacteroides sp. family, the bacteria inhabiting mammalian intestines.
Shedova
E N
EN
Berezina
O V
OV
Lunina
N A
NA
Zverlov
V V
VV
Schwartz
W H
WH
Velikodvorskaia
G A
GA
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics*; D001439:Bacteroides / Q000201:enzymology / Q000235:genetics*; D002417:Cattle; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics*; D043364:Endo-1,4-beta Xylanases / Q000235:genetics*; D023281:Genomics
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D043364:Endo-1,4-beta Xylanases