Cloning and characterisation of a large metagenomic DNA fragment containing glycosyl-hydrolase genes

Cloning and characterisation of a large metagenomic DNA fragment containing glycosyl-hydrolase genes

E. N. Shedova, O. V. Berezina, N. A. Lunina, V. V. Zverlov, W. H. Schwarz, G. A. Velikodvorskaya

ABSTRACT: The problem of searching for and characterizing enzymes produced by uncultured microorganisms is presently settled by creating metagenomic libraries. A 6000-clone library with the average size of inserts of about 15 kb has been constructed based on total DNA isolated from cow rumen microorganisms. As a result of library screening on plates with different substrates, a clone was selected that efficiently hydrolysed 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 which were identified as glycosyl hydrolase genes. No homology of the metagenomic DNA with any sequences known microorganism genomes was revealed. The amino acid sequence deduced from frame 4 was denoted of Xyl3A, and bears resemblance with β-xylosidases of glycosyl hydrolase family 3. Frame 6 encodes polypeptide Cel5A homologous to cellulases of glycosyl hydorlase family 5. The amino acid sequences deducted from on seven out of ten open reading frames were homologous to proteins of microorganisms belonging to the Bacteroides sp. family and the bacteria inhabiting mammalian intestines.

Molecular Genetics Microbiology and Virology 05/2012; 24(1):12-16.

07.05.2012, 3213 просмотров.